Graft copolymers, their preparation and use in capillary electrophoresis

ABSTRACT

The invention relates to graft copolymers, their preparation, and compositions, such as electrophoresis separation media, containing the same; also to ultra-high molecular weight poly(N,N-dimethylacrylamide) (“poly(DMA)”) polymers, their preparation, and compositions, such as electrophoresis separation media, containing the same; and more particularly to supports, such as capillaries, containing these polymers and methods for separating biomolecules, especially polynucleotides, using capillary electrophoresis. The graft copolymers can be prepared by, e.g., grafting polyacrylamide units onto a poly(DMA) backbone. Separation media comprising such graft copolymers or ultra-high molecular weight poly(DMA) polymers yield superior performance in the analysis and separation of biomolecules by capillary electrophoresis.

This application claims the benefit of U.S. provisional application No.60/399,662 filed Jul. 29, 2002, and U.S. provisional application No.60/399,663 filed Jul. 29, 2002, the disclosure of each provisionalapplication being incorporated by reference herein in its entirety.

1. FIELD OF THE INVENTION

The invention relates generally to graft copolymers, their preparation,and electrophoresis separation compositions comprising the same; also toultra-high molecular weight poly(N,N-dimethylacrylamide) polymers, theirpreparation, and electrophoresis separation compositions comprising thesame; and more particularly to supports, such as capillaries, containingthese polymers and methods for separating biomolecules, especiallypolynucleotides, using capillary electrophoresis.

2. BACKGROUND OF THE INVENTION

The technique of capillary electrophoresis (“CE”) is a widely usedanalytical method because of several technical advantages that itprovides, namely: (i) capillaries containing a separation medium havehigh surface-to-volume ratios and dissipate heat efficiently which, inturn, permits high voltage fields to be used for rapid separations; (ii)minimal sample volume is needed; (iii) superior resolution isattainable; and (iv) the technique can easily be automated, e.g.,Camilleri, Ed., Capillary Electrophoresis: Theory and Practice (CRCPress, Boca Raton, 1993); Grossman et al., Eds., CapillaryElectrophoresis (Academic Press, San Diego, 1992). Because of theseadvantages, there has been great interest in applying CE to theseparation of biomolecules, particularly in nucleic acid analysis. Theneed for rapid and accurate separation of nucleic acids, particularlydeoxyribonucleic acid (“DNA”) arises in the analysis of polymerase chainreaction products and DNA sequencing fragment analysis, e.g., Williams,Methods 4:227-232 (1992); Drossman et al., Anal. Chem., 62:900-903(1990); Huang et al., Anal. Chem., 64:2149-2154 (1992); Swerdlow et al.,Nucleic Acids Research, 18:1415-1419 (1990).

The citation of any reference in Section 2 of this application is not anadmission that the reference is prior art to the application.

3. SUMMARY OF THE INVENTION

A first embodiment of the invention relates to poly(M₁-g-M₂) or a saltthereof, where:

-   (a) each M₁ has the formula (I):

-   -   where each A₁ is independently O, S or NX₁;    -   each of R₁, R₂, R₃ and R₄ is independently H, C₁-C₂₀ alkyl,        C₄-C₁₂ cycloalkyl, C₅-C₁₂ aryl, C₄-C₁₂ heteroaryl, —(C₁-C₂₀        alkyl)(C₅-C₁₂ aryl) or —(C₅-C₁₂ aryl)(C₁-C₂₀ alkyl);    -   each R₅ is independently C₁-C₂₀ alkyl, C₁-C₂₀ heteroalkyl,        C₄-C₁₂ cycloalkyl, C₄-C₁₂ heterocycloalkyl, C₅-C₁₂ aryl, C₄-C₁₂        heteroaryl, —(C₁-C₂₀ alkyl)(C₄-C₁₂ cycloalkyl), —(C₄-C₁₂        cycloalkyl)(C₁-C₂₀ alkyl), —(C₁-C₂₀ heteroalkyl)(C₄-C₁₂        cycloalkyl), —(C₄-C₁₂ cycloalkyl)(C₁-C₂₀ heteroalkyl), —(C₁-C₂₀        alkyl)(C₄-C₁₂ heterocycloalkyl), —(C₄-C₁₂        heterocycloalkyl)(C₁-C₂₀ alkyl), —(C₁-C₂₀ heteroalkyl)(C₄-C₁₂        heterocycloalkyl), —(C₄-C₁₂ heterocycloalkyl)(C₁-C₂₀        heteroalkyl), —(C₁-C₂₀ alkyl)(C₅-C₁₂ aryl), —(C₅-C₁₂        aryl)(C₁-C₂₀ alkyl), —(C₁-C₂₀ heteroalkyl)(C₅-C₁₂ aryl),        —(C₅-C₁₂ aryl)(C₁-C₂₀ heteroalkyl), —(C₁-C₂₀ alkyl)(C₄-C₁₂        heteroaryl), —(C₄-C₁₂ heteroaryl)(C₁-C₂₀ alkyl), —(C₁-C₂₀        heteroalkyl)(C₄-C₁₂ heteroaryl), —(C₄-C₁₂ heteroaryl)(C₂-C₂₀        heteroalkyl), —(C₁-C₄ alkyl)_(q)NH₂, —(C₁-C₄ alkyl)_(q)CONH₂,        —(C₁-C₄ alkyl)NHCONH₂, —(C₁-C₄ alkyl)NHCOH or —(C₁-C₄        alkyl)_(q)NHCOCH₃, where each q is 0 or 1; and    -   each X₁ is independently H, C₁-C₂₀ alkyl, C₄-C₁₂ cycloalkyl,        C₅-C₁₂ aryl, C₄-C₁₂ heteroaryl, —(C₁-C₂₀ alkyl)(C₅-C₁₂ aryl),        —(C₅-C₁₂ aryl)(C₁-C₂₀ alkyl), —(C₁-C₄ alkyl)_(q)NH₂, —(C₁-C₄        alkyl)_(q)CONH₂, —(C₁-C₄ alkyl)NHCONH₂, —(C₁-C₄ alkyl)_(q)NHCOH        or —(C₁-C₄ alkyl)_(q)NHCOCH₃, where each q is 0 or 1;

-   (b) each M₂ has the formula (II):

-   -   where each A₂ is independently O, S or NX₂;    -   each of R₆, R₇, R₈ and R₉ is independently H, C₁-C₂₀ alkyl,        C₄-C₁₂ cycloalkyl, C₅-C₁₂ aryl, C₄-C₁₂ heteroaryl, —(C₁-C₂₀        alkyl)(C₅-C₁₂ aryl) or —(C₅-C₁₂ aryl)(C₁-C₂₀ alkyl);    -   each R₁₀ is independently H, C₁-C₂₀ alkyl, C₁-C₂₀ heteroalkyl,        C₄-C₁₂ cycloalkyl, C₄-C₁₂ heterocycloalkyl, C₅-C₁₂ aryl, C₁-C₁₂        heteroaryl, —(C₁-C₂₀ alkyl)(C₄-C₁₂ cycloalkyl), —(C₄-C₁₂        cycloalkyl)(C₁-C₂₀ alkyl), —(C₁-C₂₀ heteroalkyl)(C₄-C₁₂        cycloalkyl), —(C₄-C₁₂ cycloalkyl)(C₁-C₂₀ heteroalkyl), —(C₁-C₂₀        alkyl)(C₄-C₁₂ heterocycloalkyl), —(C₄-C₁₂        heterocycloalkyl)(C₁-C₂₀ alkyl), —(C₁-C₂₀ heteroalkyl)(C₄-C₁₂        heterocycloalkyl), —(C₄-C₁₂ heterocycloalkyl)(C₁-C₂₀        heteroalkyl), —(C₁-C₂₀ alkyl)(C₅-C₁₂ aryl), —(C₅-C₁₂        aryl)(C₁-C₂₀ alkyl), —(C₁-C₂₀ heteroalkyl)(C₅-C₁₂ aryl),        —(C₅-C₁₂ aryl)(C₁-C₂₀ heteroalkyl), —(C₁-C₂₀ alkyl)(C₄-C₁₂        heteroaryl), —(C₄-C₁₂ heteroaryl)(C₁-C₂₀ alkyl), —(C₁-C₂₀        heteroalkyl)(C₄-C₁₂ heteroaryl), —(C₄-C₁₂ heteroaryl)(C₁-C₂₀        heteroalkyl), —(C₁-C₄ alkyl)_(q)NH₂, —(C₁-C₄ alkyl)_(q)CONH₂,        —(C₁-C₄ alkyl)NHCONH₂, —(C₁-C₄ alkyl)NHCOH or —(C₁-C₄        alkyl)_(q)NHCOCH₃, where each q is 0 or 1; and    -   each X₂ is independently H, C₁-C₂₀ alkyl, C₄-C₁₂ cycloalkyl,        C₅-C₁₂ aryl, C₄-C₁₂ heteroaryl, —(C₁-C₂₀ alkyl)(C₅-C₁₂ aryl),        —(C₅-C₁₂ aryl)(C₁-C₂₀ alkyl), —(C₁-C₄—(C₁-C₄ alkyl)_(q)CONH₂,        —(C₁-C₄ alkyl)NHCONH₂, —(C₁-C₄ alkyl)_(q)NHCOH or —(C₁-C₄        alkyl)_(q)NHCOCH₃, where each q is 0 or 1;

-   (c) provided that at least one M₁ is different from at least one M₂    and that at least one A₁ or A₂ is not O.

A second embodiment of the invention relates to ultra-high molecularweight poly(N,N-dimethylacrylamide) where the weight-average molecularweight of the poly(N,N-dimethylacrylamide) is at least about 3 MDaltons.

A third embodiment of the invention relates to a method for makingultra-high molecular weight poly(N,N-dimethylacrylamide), comprising thestep of polymerizing N,N-dimethylacrylamide in an inverse emulsioncomprising an oil phase, an aqueous phase, a surfactant and aninitiator, to provide poly(N,N-dimethylacrylamide) with a weight-averagemolecular weight of at least about 3 MDaltons.

A fourth embodiment of the invention relates to a method for makingpoly(M₁-g-M₂) comprising the step of polymerizing M₂ in the presence ofan M₁ backbone polymer, i.e., “poly(M₁),” to provide poly(M₁-g-M₂).

A fifth embodiment of the invention relates to a composition comprisingpoly(M₁-g-M₂) or a salt thereof and a buffer.

A sixth embodiment of the invention relates to a composition comprisingultra-high molecular weight poly(N,N-dimethylacrylamide) and a buffer.

A seventh embodiment of the invention relates to a method for making acomposition of the invention, comprising contacting poly(M₁-g-M₂) or asalt thereof with a buffer.

An eighth embodiment of the invention relates to a method for making acomposition of the invention, comprising contacting ultra-high molecularweight poly(N,N-dimethylacrylamide) with a buffer.

A ninth embodiment of the invention relates to a method for separating amixture of biomolecules, comprising:

-   (a) contacting a composition of the invention with a mixture    comprising a biomolecule; and-   (b) applying an electric field to the composition in an amount    sufficient to facilitate the separation of a biomolecule from the    mixture.

A tenth embodiment of the invention relates to a capillary containing acomposition of the invention.

These and other objects, features and advantages of the presentinvention will become better understood with reference to the followingdescription and drawing.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the CE performance of composition IC3 in DNA sequencing.

5. DETAILED DESCRIPTION OF THE INVENTION

The invention relates generally to graft copolymers, their preparation,and compositions comprising the same; also to ultra-high molecularweight poly(N,N-dimethylacrylamide) polymers, their preparation, andcompositions comprising the same; and more particularly to supports,such as capillaries, containing these polymers and methods forseparating biomolecules, especially polynucleotides, using capillaryelectrophoresis. The graft copolymers are surprisingly and unexpectedlyeffective in, e.g., electrophoresis separation media and for use in CEas dynamic coating polymers that effectively suppress electroosmosis orelectroosmotic flow (“EOF”), which refers to capillary fluid flowinduced by an electrical field. An exemplary graft copolymer of theinvention can be described as a poly(DMA) (“PDMA”) backbone polymerbearing polyacrylamide (“PAAm”) side-chains or pendant chains.

As used herein, a “copolymer” includes a polymer comprising at least twodifferent monomeric subunits. Thus, a polymeric chain made up of threedifferent monomers (also known as a terpolymer) is included within theterm “copolymer,” as are polymer chains containing more than threedifferent monomeric units. Copolymers may be formed in many ways knownto those of ordinary skill in the art, for example: by polymerizing twodifferent monomers; by block copolymerization; by graftcopolymerization, e.g., where an existing polymer chain is furtherreacted with a different monomer; and by a post-polymerization reaction,e.g., where a polymer with ester side groups is partially hydrolyzed. Asused herein, the term “polymer” includes a homopolymer and a copolymer.

It is conventional in the polymer art that graft copolymers are commonlynamed as “poly(M₁-g-M₂),” where M₁ refers to the monomer or monomersmaking up the backbone polymer or backbone copolymer, i.e., poly(M₁);and M₂ (following the “g” for graft) refers to the monomer or monomersmaking up the grafted polymer or grafted copolymer, i.e., “poly(M₂),”sometimes also referred to herein as the pendant, pendant polymer,pendant chain or side-chain polymer. It is to be understood thatpoly(M₁) and poly(M₂) can be the same or different homopolymer orcopolymer. See G. Odian, Principles of Polymerization, McGraw-Hill BookCo., New York, 1970, pp. 366, 633 and U.S. Pat. No. 6,319,976 B1 toDeNicola, Jr. et al. for a further explanation and examples of thisnomenclature.

For example, poly(N,N-dimethylacrylamide-g-acrylamide) denotes a graftcopolymer where the backbone polymer is poly(N,N-dimethylacrylamide) andthe pendant polymer is poly(acrylamide),poly((N,N-dimethylacrylamide-co-N,N-diethyl-methacrylamide)-g-acrylamide)denotes a graft copolymer where the backbone polymer is a copolymer ofN,N-dimethylacrylamide and N,N-diethyl-methacrylamide and the pendantpolymer is poly(acrylamide), andpoly(N,N-dimethylacrylamide-g-(acrylamide-co-N-butoxymethyl-methacrylamide-co-N-methoxymethyl-acrylamide)denotes a graft copolymer where the backbone polymer ispoly(N,N-dimethylacrylamide) and the pendant polymer is a copolymer ofacrylamide, N-butoxymethyl-methacrylamide andN-methoxymethyl-acrylamide.

5.1 Graft Copolymer Poly(M₁-g-M₂)

The first embodiment of the invention relates to graft copolymerpoly(M₁-g-M₂) or a salt thereof, where:

-   (a) each M₁ has the formula (I):

-   -   where each A₁ is independently O, S or NX₁;    -   each of R₁, R₂, R₃ and R₄ is independently H, C₁-C₂₀ alkyl,        C₄-C₁₂ cycloalkyl, C₅-C₁₂ aryl, C₄-C₁₂ heteroaryl, —(C₁-C₂₀        alkyl)(C₅-C₁₂ aryl) or —(C₅-C₁₂ aryl)(C₁-C₂₀ alkyl);    -   each R₅ is independently C₁-C₂₀ alkyl, C₁-C₂₀ heteroalkyl,        C₄-C₁₂ cycloalkyl, C₄-C₁₂ heterocycloalkyl, C₅-C₁₂ aryl, C₄-C₁₂        heteroaryl, —(C₁-C₂₀ alkyl)(C₄-C₁₂ cycloalkyl), —(C₄-C₁₂        cycloalkyl)(C₁-C₂₀ alkyl), —(C₁-C₂₀ heteroalkyl)(C₄-C₁₂        cycloalkyl), —(C₄-C₁₂ cycloalkyl)(C₁-C₂₀ heteroalkyl), —(C₁-C₂₀        alkyl)(C₄-C₁₂ heterocycloalkyl), —(C₄-C₁₂        heterocycloalkyl)(C₁-C₂₀ alkyl), —(C₁-C₂₀ heteroalkyl)(C₄-C₁₂        heterocycloalkyl), —(C₄-C₁₂ heterocycloalkyl)(C₁-C₂₀        heteroalkyl), —(C₁-C₂₀ alkyl)(C₅-C₁₂ aryl), —(C₅-C₁₂        aryl)(C₁-C₂₀ alkyl), —(C₁-C₂₀ heteroalkyl)(C₅-C₁₂ aryl),        —(C₅-C₁₂ aryl)(C₁-C₂₀ heteroalkyl), —(C₁-C₂₀ alkyl)(C₄-C₁₂        heteroaryl), —(C₄-C₁₂ heteroaryl)(C₁-C₂₀ alkyl), —(C₁-C₂₀        heteroalkyl)(C₄-C₁₂ heteroaryl), —(C₄-C₁₂ heteroaryl)(C₁-C₂₀        heteroalkyl), —(C₁-C₄ alkyl)_(q)NH₂, —(C₁-C₄ alkyl)_(q)CONH₂,        —(C₁-C₄ alkyl)NHCONH₂, —(C₁-C₄ alkyl)NHCOH or —(C₁-C₄        alkyl)_(q)NHCOCH₃, where each q is 0 or 1; and    -   each X₁ is independently H, C₁-C₂₀ alkyl, C₄-C₁₂ cycloalkyl,        C₅-C₁₂ aryl, C₄-C₁₂ heteroaryl, —(C₁-C₂₀ alkyl)(C₅-C₁₂ aryl),        —(C₅-C₁₂ aryl)(C₁-C₂₀ alkyl), —(C₁-C₄ alkyl)_(q)NH₂, —(C₁-C₄        alkyl)_(q)CONH₂, —(C₁-C₄ alkyl)NHCONH₂, —(C₁-C₄ alkyl)_(q)NHCOH        or —(C₁-C₄ alkyl)_(q)NHCOCH₃, where each q is 0 or 1;

-   (b) each M₂ has the formula (II):

-   -   where each A₂ is independently O, S or NX₂;    -   each of R₆, R₇, R₈ and R₉ is independently H, C₁-C₂₀ alkyl,        C₄-C₁₂ cycloalkyl, C₅-C₁₂ aryl, C₄-C₁₂ heteroaryl, —(C₁-C₂₀        alkyl)(C₅-C₁₂ aryl) or —(C₅-C₁₂ aryl)(C₁-C₂₀ alkyl);    -   each R₁₀ is independently H, C₁-C₂₀ alkyl, C₁-C₂₀ heteroalkyl,        C₄-C₁₂ cycloalkyl, C₄-C₁₂ heterocycloalkyl, C₅-C₁₂ aryl, C₄-C₁₂        heteroaryl, —(C₁-C₂₀ alkyl)(C₄-C₁₂ cycloalkyl), —(C₄-C₁₂        cycloalkyl)(C₁-C₂₀ alkyl), —(C₁-C₂₀ heteroalkyl)(C₄-C₁₂        cycloalkyl), —(C₄-C₁₂ cycloalkyl)(C₁-C₂₀ heteroalkyl), —(C₁-C₂₀        alkyl)(C₄-C₁₂ heterocycloalkyl), —(C₄-C₁₂        heterocycloalkyl)(C₁-C₂₀ alkyl), —(C₁-C₂₀ heteroalkyl)(C₄-C₁₂        heterocycloalkyl), —(C₄-C₁₂ heterocycloalkyl)(C₁-C₂₀        heteroalkyl), —(C₁-C₂₀ alkyl)(C₅-C₁₂ aryl), —(C₅-C₁₂        aryl)(C₁-C₂₀ alkyl), —(C₁-C₂₀ heteroalkyl)(C₅-C₁₂ aryl),        —(C₅-C₁₂ aryl)(C₁-C₂₀ heteroalkyl), —(C₁-C₂₀ alkyl)(C₄-C₁₂        heteroaryl), —(C₄-C₁₂ heteroaryl)(C₁-C₂₀ alkyl), —(C₁-C₂₀        heteroalkyl)(C₄-C₁₂ heteroaryl), —(C₄-C₁₂ heteroaryl)(C₁-C₂₀        heteroalkyl), —(C₁-C₄ alkyl)_(q)NH₂, —(C₁-C₄ alkyl)_(q)CONH₂,        —(C₁-C₄ alkyl)NHCONH₂, —(C₁-C₄ alkyl)NHCOH or —(C₁-C₄        alkyl)_(q)NHCOCH₃, where each q is 0 or 1; and    -   each X₂ is independently H, C₁-C₂₀ alkyl, C₄-C₁₂ cycloalkyl,        C₅-C₁₂ aryl, C₄-C₁₂ heteroaryl, —(C₁-C₂₀ alkyl)(C₅-C₁₂ aryl),        —(C₅-C₁₂ aryl)(C₁-C₂₀ alkyl), —(C₁-C₄ alkyl)_(q)NH₂, —(C₁-C₄        alkyl)_(q)CONH₂, —(C₁-C₄ alkyl)NHCONH₂, —(C₁-C₄ alkyl)_(q)NHCOH        or —(C₁-C₄ alkyl)_(q)NHCOCH₃, where each q is 0 or 1;

-   (c) provided that at least one M₁ is different from at least one M₂    and that at least one A₁ or A₂ is not O.

It is to be understood that each M₁ or each M₂ of poly(M₁-g-M₂) or asalt thereof need not be identical. Rather poly(M₁-g-M₂) or a saltthereof can comprise non-identical M₁ and M₂ groups, i.e., the backbone,the pendant or both can be copolymers. In other embodiments, however,each M₁ or each M₂ is identical. In certain embodiments, each M₁ andeach M₂ is identical.

In certain embodiments, for each M₁, R₁ and R₂ are H, and R₃ is H ormethyl; and for each M₂, R₆ and R₇ are H, and R₈ is H or methyl.

As used herein, the term “alkyl” refers to a straight or branchedhydrocarbon chain. Examples of alkyl groups include lower alkyl, forexample, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl,tert-butyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl,2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl,4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl,4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl,2-ethyl-1-butyl, pentyl, isopentyl, neopentyl, hexyl and the like; upperalkyl, for example, n-heptyl, n-octyl, iso-octyl, ethylhexyl, nonyl,decyl, dodecyl, octadecyl and the like. The ordinary skilled artisan isfamiliar with numerous straight, i.e., linear, and branched alkylgroups, which are within the scope of the present invention. Inaddition, such alkyl groups may also include heteroalkyl groups.

The term “heteroalkyl” in the context of the present invention broadlyrefers to an alkyl possessing in-chain, pendant and/or terminalfunctionality, as understood by those persons of ordinary skill in therelevant art. As examples of in-chain functionality may be mentioned acarbonyl group or groups (which is/are, of course, included in thecarbon count), heteroatom or heteroatoms (such as at least one oxygen,sulfur, nitrogen, phosphorous or silicon) in the chain, esters, amides,urethanes and their thio-derivatives, i.e., where at least one oxygenatom is replaced by a sulfur atom. As examples of pendant and/orterminal functionality may be mentioned hydrogen-containing groups suchas hydroxyl, amino, aldehyde, carboxyl, thio and amido, and halogen.Thus, exemplary heteroalkyl groups include butoxymethyl, dimethoxybutyl,dimethoxyethyl, 3-(trimethylammonium chloride)-propyl, trimethylbutyl,acetyl, glycolic acid methyl ester, hydroxymethyl, methoxymethyl,methoxypropyl, 2,2,2-trichloro-1-hydroxyethyl,tri(hydroxymethyl)-methyl, pentafluoroethyl, bromo, chloro, 3-iodopropyland the like.

As used herein, the term “cycloalkyl” refers to a monocyclic orpolycyclic ring comprising carbon and hydrogen atoms and having nocarbon-carbon multiple bonds. Examples of cycloalkyl groups includeC₃-C₇ cycloalkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl and cycloheptyl, adamantyl, saturated cyclic and bicyclicterpenes and the like. A cycloalkyl group can be unsubstituted orsubstituted by one or more suitable substituents.

As used herein, the term “heterocycloalkyl” refers to a cycloalkyl groupcomprising a heteroatom (such as at least one oxygen, sulfur, nitrogen,phosphorous, halogen or silicon), e.g., in the ring and/or pendantthereto. Thus, a heterocycloalkyl group can be unsubstituted orsubstituted with one or more substituents. Examples of heterocycloalkylgroups include pyrrolidinyl, pyrrolidino, piperidinyl, piperidino,piperazinyl, piperazino, morpholinyl, morpholino, thiomorpholinyl,thiomorpholino and the like.

As used herein, the term “aryl” refers to a monocyclic orpolycyclic-aromatic radical comprising a hydrocarbon ring(s) bearing asystem of conjugated double bonds. In certain embodiments, thepolycyclic-aromatic radical comprising a hydrocarbon ring(s) bearing asystem of conjugated double bonds comprises at least six π (pi)electrons. An aryl group can be unsubstituted or substituted with one ormore substituents. Examples of aryl groups include phenyl, anthacenyl,fluorenyl, indenyl, azulenyl, naphthyl, anisyl, toluoyl, xylenyl and thelike.

As used herein, the term “heteroaryl” refers to an aryl group comprisinga heteroatom (such as at least one oxygen, sulfur, nitrogen,phosphorous, halogen or silicon), e.g., in the ring and/or pendantthereto. Thus, a heteroaryl group can be unsubstituted or substitutedwith one or more substituents. Examples of heteroaryl groups includehalophenyl, nitrophenyl, hydroxyphenyl, pyridinyl, pyridazinyl,pyrimidyl, pyrazyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl,(1,2,3,)- and (1,2,4)-triazolyl, pyrazinyl, pyrimidinyl, tetrazolyl,thienyl, thiazolyl, furyl, phenyl, isoxazolyl, oxazolyl, isoquinolinyl,dihydroxyisoquinolinyl, isoquinolinonyl, quinazolinyl, quinazolinonyl,naphthalimidyl, phenanthridinonyl and the like.

As used herein, a “compound term,” e.g., -(cycloalkyl)(alkyl), broadlyrefers to a monovalent first group, here cycloalkyl, in which thevalency is derived by abstraction of a hydrogen from a carbon atom orsuitable heteroatom, where the first group is further substituted by atleast one second group, here an alkyl group(s), e.g., 3-methylcyclohexylor isobornyl. As a further illustration, a compound term such as-(alkyl)(aryl) refers to a first group, here alkyl, which is furthersubstituted with at least one second group, here an aryl group(s), e.g.,benzyl or 2,2-diphenyl ethyl.

As used herein, a “salt” of a polymer refers to a polymer having atleast one anionic charge, cationic charge, or both, e.g., an amphotericpolymer, where each charge has associated with it a suitable counterion.“Counterion” refers to an ion that balances the polymer's anionic orcationic charge. Exemplary counterions for a polymer comprising acationic charge include chloride, bromide, iodide, hydroxide, alkoxide,carbonate, bicarbonate, oxide, formate, sulfate, benzene sulfonate,p-toluenesulfonate, p-bromobenzenesulfonate, methanesulfonate,trifluoromethanesulfonate, phosphate, perchlorate, tetrafluoroborate,tetraphenylboride, nitrate and anions of aromatic or aliphaticcarboxylic acids. Exemplary counterions for a polymer comprising ananionic charge include ammonium, quaternary phosphonium, such as atetraalkyl phosphonium halide, and the cations of Li, Na, K, Rb, Cs andAg. In certain embodiments, the counterions include chloride,p-toluenesulfonate, lithium, sodium and potassium.

In an embodiment of the invention, the graft copolymer is poly(M₁-g-M₂)or a salt thereof, where:

-   (a) M₁ is N-adamantyl-acrylamide, N-butoxymethyl-acrylamide,    N,N-dibutyl-acrylamide, N,N-diethyl-acrylamide,    N-4,4-dimethoxybutyl-acrylamide, N,N-dimethyl-acrylamide,    N,N-dipropyl-acrylamide, N-dodecyl-acrylamide,    N-2-ethylhexyl-acrylamide, N-isobornyl-acrylamide, N-methyl-,    N-2,2-dimethoxyethyl-acrylamide, N-morpholinoethyl-acrylamide,    N-octadecyl-acrylamide, N-3-(trimethylammonium)-propyl-acrylamide    chloride, N-1,1,3-trimethylbutyl-acrylamide,    N-adamantyl-methacrylamide, N-butoxymethyl-methacrylamide,    N,N-dibutyl-methacrylamide, N,N-diethyl-methacrylamide,    N-4,4-dimethoxybutyl-methacrylamide, N,N-dimethyl-methacrylamide,    N,N-dipropyl-methacrylamide, N-dodecyl-methacrylamide,    N-2-ethylhexyl-methacrylamide, N-isobornyl-methacrylamide,    N-methyl-, N-2,2-dimethoxyethyl-methacrylamide,    N-morpholinoethyl-methacrylamide, N-octadecyl-methacrylamide,    N-3-(trimethylammonium)-propyl-methacrylamide chloride,    N-1,1,3-trimethylbutyl-methacrylamide, or a mixture thereof;-   (b) M₂ is acrylamide, N-acetyl-acrylamide,    N-butoxymethyl-acrylamide, N-4,4-dimethoxybutyl-acrylamide,    N-2,2-dimethoxyethyl-acrylamide, N-2-glycolic acid methyl ester    acrylamide, N-hydroxymethyl-acrylamide, N-methoxymethyl-acrylamide,    N-3-methoxypropyl-acrylamide, N-methyl-,    N-2,2-dimethoxyethyl-acrylamide, N-morpholinoethyl-acrylamide,    N-2,2,2-trichloro-1-hydroxyethyl-acrylamide,    N-tri(hydroxymethyl)-methyl-acrylamide, methacrylamide,    N-acetyl-methacrylamide, N-butoxymethyl-methacrylamide,    N-4,4-dimethoxybutyl-methacrylamide,    N-2,2-dimethoxyethyl-methacrylamide, N-2-glycolic acid methyl ester    methacrylamide, N-hydroxymethyl-methacrylamide,    N-methoxymethyl-methacrylamide, N-3-methoxypropyl-methacrylamide,    N-methyl-, N-2,2-dimethoxyethyl-methacrylamide,    N-morpholinoethyl-methacrylamide,    N-2,2,2-trichloro-1-hydroxyethyl-methacrylamide,    N-tri(hydroxymethyl)-methyl-methacrylamide, or a mixture thereof;-   (c) provided that at least one M₁ is different from at least one M₂.

In another embodiment of the invention, the graft copolymer ispoly(M₁-g-M₂) or a salt thereof, where:

-   (a) M₁ is N-adamantyl-acrylamide, N-butoxymethyl-acrylamide,    N,N-dibutyl-acrylamide, N,N-diethyl-acrylamide,    N-4,4-dimethoxybutyl-acrylamide, N,N-dimethyl-acrylamide,    N,N-dipropyl-acrylamide, N-dodecyl-acrylamide,    N-2-ethylhexyl-acrylamide, N-isobornyl-acrylamide, N-methyl-,    N-2,2-dimethoxyethyl-acrylamide, N-morpholinoethyl-acrylamide,    N-octadecyl-acrylamide, N-3-(trimethylammonium)-propyl-acrylamide    chloride, N-1,1,3-trimethylbutyl-acrylamide,    N-adamantyl-methacrylamide, N-butoxymethyl-methacrylamide,    N,N-dibutyl-methacrylamide, N,N-diethyl-methacrylamide,    N-4,4-dimethoxybutyl-methacrylamide, N,N-dimethyl-methacrylamide,    N,N-dipropyl-methacrylamide, N-dodecyl-methacrylamide,    N-2-ethylhexyl-methacrylamide, N-isobornyl-methacrylamide,    N-methyl-, N-2,2-dimethoxyethyl-methacrylamide,    N-morpholinoethyl-methacrylamide, N-octadecyl-methacrylamide,    N-3-(trimethylammonium)-propyl-methacrylamide chloride,    N-1,1,3-trimethylbutyl-methacrylamide, or a mixture thereof;-   (b) M₂ is N-acetyl-acrylamide, N-butoxymethyl-acrylamide,    N-4,4-dimethoxybutyl-acrylamide, N-2,2-dimethoxyethyl-acrylamide,    N-2-glycolic acid methyl ester acrylamide,    N-hydroxymethyl-acrylamide, N-methoxymethyl-acrylamide,    N-3-methoxypropyl-acrylamide, N-methyl-,    N-2,2-dimethoxyethyl-acrylamide, N-morpholinoethyl-acrylamide,    N-2,2,2-trichloro-1-hydroxyethyl-acrylamide,    N-tri(hydroxymethyl)-methyl-acrylamide, methacrylamide,    N-acetyl-methacrylamide, N-butoxymethyl-methacrylamide,    N-4,4-dimethoxybutyl-methacrylamide,    N-2,2-dimethoxyethyl-methacrylamide, N-2-glycolic acid methyl ester    methacrylamide, N-hydroxymethyl-methacrylamide,    N-methoxymethyl-methacrylamide, N-3-methoxypropyl-methacrylamide,    N-methyl-, N-2,2-dimethoxyethyl-methacrylamide,    N-morpholinoethyl-methacrylamide,    N-2,2,2-trichloro-1-hydroxyethyl-methacrylamide,    N-tri(hydroxymethyl)-methyl-methacrylamide, or a mixture thereof;-   (c) provided that at least one M₁ is different from at least one M₂.

In another embodiment of the invention, the graft copolymer ispoly(M₁-g-M₂) or a salt thereof, where:

-   (a) M₁ is N-adamantyl-acrylamide, N-butoxymethyl-acrylamide,    N,N-dibutyl-acrylamide, N,N-diethyl-acrylamide,    N-4,4-dimethoxybutyl-acrylamide, N,N-dimethyl-acrylamide,    N,N-dipropyl-acrylamide, N-dodecyl-acrylamide,    N-2-ethylhexyl-acrylamide, N-isobornyl-acrylamide, N-methyl-,    N-2,2-dimethoxyethyl-acrylamide, N-morpholinoethyl-acrylamide,    N-octadecyl-acrylamide, N-3-(trimethylammonium)-propyl-acrylamide    chloride, N-1,1,3-trimethylbutyl-acrylamide,    N-adamantyl-methacrylamide, N-butoxymethyl-methacrylamide,    N,N-dibutyl-methacrylamide, N,N-diethyl-methacrylamide,    N-4,4-dimethoxybutyl-methacrylamide, N,N-dimethyl-methacrylamide,    N,N-dipropyl-methacrylamide, N-dodecyl-methacrylamide,    N-2-ethylhexyl-methacrylamide, N-isobornyl-methacrylamide,    N-methyl-, N-2,2-dimethoxyethyl-methacrylamide,    N-morpholinoethyl-methacrylamide, N-octadecyl-methacrylamide,    N-3-(trimethylammonium)-propyl-methacrylamide chloride,    N-1,1,3-trimethylbutyl-methacrylamide, or a mixture thereof;-   (b) M₂ is N-acetyl-acrylamide, N-butoxymethyl-acrylamide,    N-4,4-dimethoxybutyl-acrylamide, N-2,2-dimethoxyethyl-acrylamide,    N-2-glycolic acid methyl ester acrylamide,    N-hydroxymethyl-acrylamide, N-methoxymethyl-acrylamide,    N-3-methoxypropyl-acrylamide, N-methyl-,    N-2,2-dimethoxyethyl-acrylamide, N-morpholinoethyl-acrylamide,    N-2,2,2-trichloro-1-hydroxyethyl-acrylamide,    N-tri(hydroxymethyl)-methyl-acrylamide, N-acetyl-methacrylamide,    N-butoxymethyl-methacrylamide, N-4,4-dimethoxybutyl-methacrylamide,    N-2,2-dimethoxyethyl-methacrylamide, N-2-glycolic acid methyl ester    methacrylamide, N-hydroxymethyl-methacrylamide,    N-methoxymethyl-methacrylamide, N-3-methoxypropyl-methacrylamide,    N-methyl-, N-2,2-dimethoxyethyl-methacrylamide,    N-morpholinoethyl-methacrylamide,    N-2,2,2-trichloro-1-hydroxyethyl-methacrylamide,    N-tri(hydroxymethyl)-methyl-methacrylamide, or a mixture thereof;-   (c) provided that at least one M₁ is different from at least one M₂.

Alternately or in addition to M₂, the pendant can comprisepoly(hydroxymethylene), poly(oxyethylene), poly(oxypropylene),poly(oxyethylene-co-oxypropylene), poly(vinyl alcohol),poly(vinylpyrrolidone), poly(2-ethyl-2-oxazoline),poly(2-methyl-2-oxazoline),poly((2-ethyl-2-oxazoline)-co-(2-methyl-2-oxazoline)),poly(N-acetamidoacrylamide), poly(acryloxylurea), water-solublepolysaccharides such as hydroxyethyl cellulose and hydroxymethylcellulose, or a mixture thereof.

In another embodiment, the poly(M₁-g-M₂) or a salt thereof ispoly(N-butoxymethyl-acrylamide-g-acrylamide),poly(N,N-dibutyl-acrylamide-g-acrylamide),poly(N,N-diethyl-acrylamide-g-acrylamide),poly(N-4,4-dimethoxybutyl-acrylamide-g-acrylamide),poly(N,N-dimethyl-acrylamide-g-acrylamide),poly(N,N-dipropyl-acrylamide-g-acrylamide),poly(N-2-ethylhexyl-acrylamide-g-acrylamide),poly(N-isobornyl-acrylamide-g-acrylamide),poly(N-3-(trimethylammonium)-propyl-acrylamide chloride-g-acrylamide),poly(N-1,1,3-trimethylbutyl-acrylamide-g-acrylamide),poly(N-butoxymethyl-methacrylamide-g-acrylamide),poly(N,N-dibutyl-methacrylamide-g-acrylamide),poly(N,N-diethyl-methacrylamide-g-acrylamide),poly(N-4,4-dimethoxybutyl-methacrylamide-g-acrylamide),poly(N,N-dimethyl-methacrylamide-g-acrylamide),poly(N,N-dipropyl-methacrylamide-g-acrylamide),poly(N-1,1,3-trimethylbutyl-methacrylamide-g-acrylamide),poly((N,N-dimethyl-acrylamide-co-N,N-dibutyl-acrylamide)-g-acrylamide),poly((N,N-dimethyl-acrylamide-co-N,N-dibutyl-methacrylamide)-g-acrylamide),poly((N,N-dimethyl-methacrylamide-co-N,N-dibutyl-acrylamide)-g-acrylamide),poly((N,N-dimethyl-methacrylamide-co-N,N-dibutyl-methacrylamide)-g-acrylamide),poly(N-butoxymethyl-acrylamide-g-(acrylamide-co-N-hydroxymethyl-acrylamide)),poly(N-butoxymethyl-acrylamide-g-(acrylamide-co-N-hydroxymethyl-methacrylamide)),poly(N-butoxymethyl-acrylamide-g-methacrylamide),poly(N,N-dibutyl-acrylamide-g-methacrylamide),poly(N,N-diethyl-acrylamide-g-methacrylamide),poly(N-4,4-dimethoxybutyl-acrylamide-g-methacrylamide),poly(N,N-dimethyl-acrylamide-g-methacrylamide),poly(N,N-dipropyl-acrylamide-g-methacrylamide),poly(N-2-ethylhexyl-acrylamide-g-methacrylamide),poly(N-isobornyl-acrylamide-g-methacrylamide),poly(N-3-(trimethylammonium)-propyl-acrylamidechloride-g-methacrylamide),poly(N-1,1,3-trimethylbutyl-acrylamide-g-methacrylamide),poly(N-butoxymethyl-methacrylamide-g-methacrylamide),poly(N,N-dibutyl-methacrylamide-g-methacrylamide),poly(N,N-diethyl-methacrylamide-g-methacrylamide),poly(N-4,4-dimethoxybutyl-methacrylamide-g-methacrylamide),poly(N,N-dimethyl-methacrylamide-g-methacrylamide),poly(N,N-dipropyl-methacrylamide-g-methacrylamide),poly(N-1,1,3-trimethylbutyl-methacrylamide-g-methacrylamide),poly((N,N-dimethyl-acrylamide-co-N,N-dibutyl-acrylamide)-g-methacrylamide),poly((N,N-dimethyl-acrylamide-co-N,N-dibutyl-methacrylamide)-g-methacrylamide),poly(N,N-dimethyl-methacrylamide-co-N,N-dibutyl-acrylamide)-g-methacrylamide),poly((N,N-dimethyl-methacrylamide-co-N,N-dibutyl-methacrylamide)-g-methacrylamide),poly(N-butoxymethyl-acrylamide-g-(methacrylamide-co-N-hydroxymethyl-acrylamide)),orpoly(N-butoxymethyl-acrylamide-g-(methacrylamide-co-N-hydroxymethyl-methacrylamide)).

In one embodiment, M₁ is N,N-dimethylacrylamide and M₂ is acrylamide.

In another embodiment, a graft copolymer of the invention is watersoluble, water swellable or both, at atmospheric pressure, aconcentration of from about 0.01 to about 1 wt. %, and from about 20° C.to about 70° C., e.g., at 25° C. For purposes of this invention, waterswellable graft copolymers are generally either those that swell inwater but appear not to be completely soluble because they have a veryslow dissolution rate, e.g., graft copolymers that are substantiallyuncrosslinked but have an extremely high weight-average molecularweight; or those unable to dissolve completely in water because theyhave been crosslinked to a certain low degree, for example, bysynthesizing the copolymer to comprise certain amounts of crosslinkingor branching agents. In one embodiment, a graft copolymer of theinvention is substantially uncrosslinked such that it is able to flowinto or out of a capillary either with or without the assistance ofpressure or vacuum. In another embodiment, a graft copolymer of theinvention is substantially uncrosslinked by covalent chemical bonds.

The weight-average molecular weight (“Mw”) of the backbone polymer canvary widely. In one embodiment, the backbone polymer Mw is from about50,000 Daltons (“Da”). In another embodiment, the backbone polymer Mw isfrom about 75,000 Da, to about 15 MegaDaltons (“MDa”). In anotherembodiment, the Mw of the backbone polymer is from about 100,000 Da toabout 10 MDa. In another embodiment, the Mw of the backbone polymer isfrom about 500,000 Da to about 8 MDa. In another embodiment, the Mw ofthe backbone polymer is from about 800,000 Da to about 5 MDa.

The ratio of the pendant/backbone by weight, i.e., the sum of the weightof all M₂ units present in poly(M₁-g-M₂) divided by the sum of theweight of all M₁ units present, can vary widely. In one embodiment, theratio of the pendant/backbone is from about 0.01 to about 500. Inanother embodiment, the ratio of the pendant/backbone is from about 0.05to about 200. In another embodiment, the ratio of the pendant/backboneis from about 0.1 to about 20 by weight.

The Mw of the poly(M₁-g-M₂) can vary widely. In one embodiment, the Mwof the poly(M₁-g-M₂) is from about 150,000 Da to about 20 MDa. Inanother embodiment, the Mw of the poly(M₁-g-M₂) is from about 500,000 Dato about 10 MDa. In another embodiment, the Mw of the poly(M₁-g-M₂) isfrom about 1 MDa to about 6 MDa.

The conventional method of gel permeation chromatography (“GPC”), alsoknown as size exclusion chromatography or SEC, is a reliable way fordetermining the molecular weight of polymers and copolymers. Thefundamentals of applying a multi-angle laser light scattering detectorto GPC instrumentation (“GPC-MALLS”) for the absolute characterizationof polymers, such as the determination of their number-average andweight-average molecular weights, are conventional, e.g, see Wyatt,Analytica Chimica Acta 272:1-40 (1993). For example, GPC-MALLS has beenused to determine, inter alia, the Mw of several water-soluble polymersand copolymers with molecular weights of from below 50,000 Da to over 1MDa. Nagy, Proc. Int'l. GPC Symposium, Orlando, Fla., June 199495-0315:71-95 (1994). Moreover, the accuracy of GPC and, in particular,GPC-MALLS, for molecular weight determinations is well-recognized in theart. For example, the GPC molecular weight results obtained foridentical polymer samples using several different advanced on-linedetection systems, including GPC-MALLS compare favorably. S. Yau,Chemtracts—Macromolecular Chemistry 1:1-36 (1990). Therefore, GPC-MALLSis a convenient way for determining the number-average andweight-average molecular weights of poly(M₂), poly(M₂) and poly(M₁-g-M₂)polymers of the invention.

5.2 Method for Making Graft Copolymer

Many methods of making graft copolymers are known in the art and can beused to prepare the poly(M₁-g-M₂) of the present invention. For example,several such methods are summarized in the following chapter: Costelloet al., “Copolymers” in Kirk-Othmer Encyc. of Chem. Technol., 4th Ed.,John Wiley & Sons, New York, 1993, Vol. 7, pp. 349-381. Theseconventional methods include selecting a polymeric backbone withsuitable reactive sites, selecting a monomer or monomers, e.g., M₂ ofthe invention, and then conducting a polymerization, e.g., initiated byfree-radical, anionic or cationic means, of that monomer(s) to form agraft copolymer. Id., pp. 356-358. Such polymerizations can, of course,be conducted in bulk, solution, suspension, emulsion or microemulsion,and a wide variety of polymerization initiators can be used. Id., p.356.

Many types of free-radical initiators are suitable, e.g., azo and diazocompounds, such as azo-bis-isobutyronitrile (“AIBN”), organic peroxides,hydroperoxides, persulfates and hydropersulfates, such as benzoylperoxide, inorganic peroxides and persulfates, such as theperoxide-redox systems, carbon-carbon initiators, such ashexasubstituted ethanes, and photoinitiators; numerous examples areknown in the art. See Sanchez et al., “Initiators (Free-Radical)” inKirk-Othmer Encyc. of Chem. Technol., 4th Ed., John Wiley & Sons, NewYork, 1995, Vol. 14, pp. 431-460. Suitable anionic initiators are knownin the art and include aromatic radical anions, such as sodiumnaphthalene; alkyl lithium compounds, such as t-butyl lithium; fluorenylcarbanions; 1,1-diphenylmethylcarbanions; cumyl potassium; and thosedescribed by Quirk et al., “Initiators (Anionic)” in Kirk-Othmer Encyc.of Chem. Technol., 4th Ed., John Wiley & Sons, New York, 1995, Vol. 14,pp. 461-476. Suitable cationic initiators are also known in the art andinclude protic acids, cation donor (initiator)/Friedel-Crafts acid(coinitiator) systems, stable cation salts, and those described byFaust, “Initiators (Cationic)” in Kirk-Othmer Encyc. of Chem. Technol.,4th Ed., John Wiley & Sons, New York, 1995, Vol. 14, pp. 476-482. Thefree-radical, anionic or cationic initiator may undergo decomposition byany known means, e.g., thermally or photolytically, when this isrequired to initiate polymerization.

A suitable backbone polymer, such as poly(N,N-dimethylacrylamide)(“PDMA”), can be synthesized using techniques known in the art, e.g., asdisclosed in Trossarelli et al., J. Polymer Sci., 57:445-452 (1962) andin U.S. Pat. No. 5,916,426 to Madabhushi et al.

In particular, eight examples of the free-radical initiated synthesis ofPDMA in dioxane via solution-polymerization are disclosed in Example 1of the latter, at col. 10, line 40 to col. 11, line 12, which is herebyincorporated by reference. The following representative procedure issuitably useful for forming satisfactory PDMA. From 5 to 70% g DMA percc dioxane and from 1.2 to 16.4 mg of the free-radical initiator AIBN/gDMA are mixed at about 25° C. in an Erlenmeyer flask and argon gas isbubbled through the solution for 10 minutes at room temperature. DMApolymerization is initiated by raising the temperature to about 55° C.Polymerization times of from about 10 to about 25 minutes aresatisfactory, depending upon the concentration of monomer. Afterpolymerization, each of the polymers formed may be purified, e.g., bythree cycles of precipitation in hexane and dissolution in methylenechloride. Finally, the hexane precipitate may be dried overnight in avacuum desiccator and then lyophilized. The Mw of the PDMA productobtained, which may be conveniently determined using GPC, should be inthe range of from about 54,000 to about 99,000 Da.

Additionally, four examples of the free-radical initiated synthesis ofPDMA in tert-butyl alcohol via solution-polymerization are disclosed inExample 2 of U.S. Pat. No. 5,916,426, at col. 11, lines 13-35, which ishereby incorporated by reference. The tert-butyl alcohol acts not onlyas a solvent but as a chain transfer agent that reduces the PDMAmolecular weight. The following representative procedure isalternatively useful for forming satisfactory PDMA. From 50 to 70% g DMAper cc tert-butyl alcohol and from about 1.2 to 1.7 mg of AIBN/g DMA aremixed at about 25° C. in an Erlenmeyer flask and argon gas is bubbledthrough the solution for about 20 minutes. DMA polymerization isinitiated as described above and is allowed to continue for about 15minutes. The resulting polymers may be isolated as described above andlyophilized to yield a PDMA product with a Mw of from about 81,000 toabout 112,000 Da.

Those skilled in the art will, of course, recognize that the PDMA Mw canbe controlled by conventional methods, e.g., substituting with anotherchain transfer agent, such as n-butanol or isopropanol, with a differentchain transfer constant to monomer and/or varying the amount of chaintransfer agent present. In particular, the Mw can be increased bydecreasing the initiator concentration relative to the starting monomerconcentration and/or decreasing the amount of chain transfer agentpresent, or even eliminating the chain transfer agent entirely.

The Mw of the PDMA backbone can vary widely. In one embodiment, the Mwof the PDMA backbone is from about 50,000 Da to less than about 3 MDawhen the PDMA is prepared by solution-polymerization. In anotherembodiment, the Mw of a solution-polymerized PDMA is from about 75,000Da to about 2 MDa. As the target PDMA Mw increases, e.g., beginning atabout 2 MDa and higher, the difficulty of solution-polymerizationincreases greatly because the solution becomes excessively viscous whichmay lead to problems with, e.g., homogeneity, poor heat transfer andease of mixing. For PDMAs having a Mw greater than about 3 MDa, othermethods, including those described below, may be more effective. As usedherein, high weight-average molecular weight (“HMw”) polymers and,particularly, HMw PDMA polymers, include those with a Mw of up to lessthan about 3 MDa. As used herein, ultra-high weight-average molecularweight (“UHMw”) polymers and, particularly, UHMw PDMA polymers, includesthose with a Mw of greater than 3 MDa. Thus, PDMA backbone polymersprepared by conventional solution-polymerization methods are limited tothe HMw PDMAs.

5.3 Inverse Emulsion Polymerization of DMA to UHMw PDMA

Surprisingly, it has been found that UHMw PDMA may be prepared by themethod of inverse emulsion polymerization (“IEP”). Many aspects of theIEP method have been described in detail by, e.g., “Inverse Emulsion(Microemulsion) Polymerization,” Chapter 4 in Radical Polymerization inDisperse Systems, Barton et al., Ellis Horwood, New York, 1994, pp.187-215; Candau et al., J. Polym. Sci., Polym. Chem. Ed., 23:193-214(1985); and Pross et al., Polym. Intl., 45:22-26 (1998). IEP issometimes referred to as inverse microsuspension polymerization (Pross,p. 22.) or as inverse microemulsion polymerization (Barton, Id.).However, the preparation of UHMw PDMA by the IEP method has not beenpreviously known, described or suggested.

Any suitable oil can be used to form the inverse emulsion. To producedesirable UHMw PDMA from DMA, the DMA should be present in the waterphase. Without being bound by a particular theory, because DMA ispartially soluble in the oils commonly used in the art as the oil phasefor IEP, its oil-solubility is thought to limit the maximum molecularweight of the polymer produced when using such oils. Thus, when UHMwpolymers are to be made, it is desirable that their monomer(s) besubstantially insoluble in the oil selected.

For the purpose of selecting an appropriate monomer/oil combination,“oil insoluble” is defined by the following uncomplicated test. At atemperature of 20° C. throughout, 1 mL of the selected monomer ormonomer mixture is placed into 6 mL of the selected oil(s) and vortexmixed for 1 minute. The mixing is stopped and the liquid is allowed tostand for 10 minutes. The monomer(s) is insoluble in the oil(s) if phaseseparation, e.g., translucency, cloudiness and/or separate layers, canbe observed with the unaided eye after the 10 minute period. Conversely,the monomer(s) is soluble in the oil(s) if no phase separation, i.e., aclear solution, is observed.

For example, by this test DMA was determined to be soluble in each ofacetonitrile, acetone, methanol, 1-decanol, ethyl ether, hexane, decane,petroleum ether (normal boiling range 35-60° C.), and petroleum special(normal boiling range 180-220° C.). Therefore, none of these materials,individually, would be a preferable oil phase for forming UHMw PDMA fromDMA by IEP. However, DMA was determined to be insoluble by this test in,e.g., aliphatic hydrocarbons comprising at least about 15 carbon atoms.Alternatively, DMA was determined to be insoluble by this test in, e.g.,aliphatic hydrocarbons with a normal boiling point at or above about270° C. Exemplary hydrocarbons that are suitable oils for forming UHMwPDMA by IEP from DMA include pentadecane, hexadecane, heptadecane, whitelight mineral or paraffin oils, white heavy mineral or paraffin oils,and mineral or paraffin oils suitable for Nujol preparations.

DMA is also insoluble by the above test in, e.g., silicone oils, atleast partially fluorinated hydrocarbons and liquid perfluoropolyethers(“PFPE”), also known as perfluoropolyalkylethers (“PFPAE”).

Exemplary silicones that are conventional and suitable oils for formingUHMw PDMA by IEP from DMA include poly(dimethylsiloxane)-based oils suchas DC200, DC510, DC550 and DC710, each of which may be available invarious viscosity grades (e.g., from 10 cSt to 12,500 cSt for DC200)from Dow Corning and poly(methylphenylsiloxane)-based oils such asAR200, also from Dow Corning.

Exemplary at least partially fluorinated hydrocarbon liquids that areconventional and suitable oils for forming UHMw PDMA by IEP from DMAinclude the FLUORINERT series available from 3M, e.g., FC-40, FC-43,FC-70, FC-72, FC-77, FC-84, FC-87, FC-3283, FC-5312 and FC-5320

Exemplary liquid PFPEs that are conventional and suitable oils forforming UHMw PDMA by IEP from DMA include the DEMNUIM series availablefrom Daikin Industries, Ltd., e.g., S-20, S-65, S-100 and S-200, theKRYTOX series available from DuPont, e.g., GPL100, GPL101, GPL102,GPL103, GPL104, GPL105, GPL106, GPL107, 143AB, 143AC and VPF1525, andthe FOMBLIN Y, Z and M series available from Ausimont Montedison Group,e.g., Y04, Y06, Y25, Y-L VAC 25/6, YR, YR1500, YR1800, Z03, Z15, Z25,Z60, M03, M15, M30 and M60. As disclosed by, e.g., Hamada, Phys. Chem.Chem. Phys., 2:115-122 (2000), the DEMNUIM-type PFPEs have the formulaF—[CF₂CF₂CF₂O]_(n)—H, the KRYTOX-type PFPEs have the formulaF—[CF(CF₃)CF₂O], —H, and the FOMBLIN-Z-type PFPEs have the formulaF—[(CF₂CF₂O)₂—(CF₂O)]_(m)—H, where n, l and m are varied to give, e.g.,different chain lengths and viscosities.

In one embodiment, oils for the IEP of DMA to UHMw PDMA includealiphatic hydrocarbons comprising at least about 15 carbon atoms,aliphatic hydrocarbons with a normal boiling point at or above about270° C., silicone oils, at least partially fluorinated hydrocarbons,liquid perfluoropolyethers, or a mixture thereof. In another embodiment,oils for the IEP of DMA to UHMw PDMA include pentadecane, hexadecane,heptadecane, white light mineral oils, white heavy mineral oils, andmineral oils suitable for Nujol preparations. In another embodiment, theoil used for the IEP of DMA to UHMw PDMA is a mineral oil suitable forNujol preparations.

At least one surfactant is used to form the inverse emulsion. When aplurality of surfactants is present, the additional surfactant(s) issometimes known as a cosurfactant. It is conventional to characterize asurfactant by its hydrophilic lipophilic balance (“HLB”), a measure ofthe relative simultaneous attraction of the surfactant for water andoil. On the HLB scale ranging from 1 to 40, relatively lipophilicsurfactants have a low numerical value while relatively hydrophylicsurfactants have a high numerical value.

A wide variety of surfactants are known to be available, for example,many are listed with HLB values in McCutcheon's Emulsifiers &Detergents, North American Ed., Manufacturing Confectioner Pub. Co.,Glen Rock, N.J., 1988, pp. 1-217. The surfactant may be nonionic or havean anionic charge, cationic charge, or both, e.g., an amphotericsurfactant, where each charge has associated with it a suitablecounterion; numerous examples of each are known in the art. See Lynn,Jr. et al., “Surfactants” in Kirk-Othmer Encyc. of Chem. Technol., 4thEd., John Wiley & Sons, New York, 1997, Vol. 23, pp. 483-541.

Suitable types of nonionic surfactants are known in the art and includepolyoxyethylene surfactants, e.g., alcohol ethoxylates and alkylphenolethoxylates; carboxylic acid esters, e.g., glycerol esters andpolyoxyethylene esters; anhydrosorbitol esters, e.g., mono-, di- andtri-esters of sorbitan and fatty acids; polyalkylene oxide blockcopolymers; and poly(oxyethylene-co-oxypropylene) nonionic surfactants.Id., pp. 506-523.

Suitable types of anionic surfactants are known in the art and includecarboxylates, e.g., soaps, polyalkoxycarboxylates andN-acylsarcosinates; sulfonates, e.g., alkylbenzene sulfonates,naphthalene sulfonates and petroleum sulfonates; sulfates, e.g., alcoholsulfates and ethoxylated and sulfated alcohols; and phosphates, e.g.,phosphate esters. Id., pp. 491-505.

Suitable types of cationic surfactants are known in the art and includeamines, e.g., aliphatic mono-, di- and polyamines derived from fatty androsin acids; and quaternary ammonium salts, e.g., dialkyldimethyl andalkyltrimethyl ammonium salts, alkylbenzyldimethyl ammonium chlorides,and alkylpyridinium halides. Id., pp. 524-530. Suitable types ofamphoteric surfactants are known in the art and include alkylbetaines,amidopropylbetaines and imidazolinium derivatives. Id., pp. 530-532.

Considerations typically taken into account in selecting a suitablesurfactant or surfactant blend to form an inverse emulsion areconventional and are summarized in, e.g., Griffin, “Emulsions” inKirk-Othmer Encyc. of Chem. Technol., 3rd Ed., John Wiley & Sons, NewYork, 1979, Vol. 8, pp. 909-919. Furthermore, it is recognized that somemonomers, e.g., acrylamide, can sometimes act as a co-surfactant.Candau, p. 204; Barton, p. 191. In such cases, the overall HLB value ofthe emulsification system can differ from the HLB of the selectedsurfactant or surfactant blend. Barton, p. 191. Moreover, those in theart would recognize that particular characteristics of the inverseemulsion must be taken into account when selecting a surfactant. Forexample, when a fluorinated oil is used, it is desirable to also selectan at least partially fluorinated surfactant. Taking such conventionalfactors into consideration, one of ordinary skill in the art would beable to select a wide variety of suitable surfactants, used individuallyor in combination, for the IEP of water-soluble monomers to form UHMwpolymers and, particularly, to form UHMw PDMA from DMA by IEP.

In one embodiment, surfactants for the IEP of DMA to UHMw PDMA have anHLB of about 7 or less. In another embodiment, the surfactant HLB isabout 6 or less. In another embodiment, the surfactant HLB is from about3 to about 6. In another embodiment, surfactants for the IEP of DMA toUHMw PDMA have an HLB of from about 4 to about 6. In another embodiment,surfactants include SPAN-80 from Fluka, sorbitan monooleate thought tohave the following structure:

with a molecular weight of about 429 Da and an HLB of about 4.3;TETRONIC 1301 from BASF, an amine-based block copolymer nonionicsurfactant thought to have the following structure:

with a molecular weight of about 6,800 Da and an HLB of about 2.0; or amixture thereof.

A sufficient amount of the surfactant is used such that a stableemulsion or microemulsion is formed; routine experimentation by oneordinarily-skilled in the art can be used to determine that amount. Toobtain, after polymerization, a microemulsion of high polymer content,the ratio (by weight) of aqueous phase to oil phase is usually chosen tobe as high as possible. This ratio may range, for example, from about1:10 to about 4:1. In another embodiment, the ratio may range from about1:2 to about 3:1. In another embodiment, the quantity of solid polymerproduct is greater than about 10 wt. % of the total emulsion weight.

Many types of initiators discussed above are also suitable for use ininverse emulsion polymerizations, e.g., free-radical initiators such asthe azo compounds, organic peroxides and persulfates, inorganicperoxides and persulfates, and carbon-carbon initiators, as well asphotoinitiators such as those described in McGinniss, “Radiation Curing”in Kirk-Othmer Encyc. of Chem. Technol., 4th Ed., John Wiley & Sons, NewYork, 1996, Vol. 20, pp. 848-850. Polymerization may, of course, also beeffected by high energy ionizing radiation sources.

In one embodiment, inverse emulsion polymerization initiators includethe azo compounds, either the oil-soluble types such as AIBN or thewater-soluble types such as azobutyroamidine, oil-soluble peroxides andpersulfates, such as dibenzoyl peroxide, water soluble peroxides andpersulfates, such as ammonium persulfate and potassium persulfate, redoxinitiating systems, which include the peroxy-redox types and, e.g.,K₂S₂O₈/Na₂S₂O₅ or ferrous ammonium sulfate/ammonium persulfate, andphotoinitiators, such as Michler's ketone, i.e.,4,4′-bis-(dimethylamino)benzophenone, and IRGACURE-1700 andDAROCURE-1173 from Ciba-Geigy, believed to be (25%bis(2,6-dimethoxybenzoyl)-2,4,4-trimethylpentylphosphine oxide+75%2-hydroxy-2-methyl-1-phenyl-propan-1-one) and2-hydroxy-2-methyl-1-phenyl-propan-1-one, respectively. In anotherembodiment, initiators for the IEP of DMA to UHMw PDMA includeoil-soluble azo compounds, water soluble peroxides and persulfates,redox initiating systems, photoinitiators, or a mixture thereof. Inanother embodiment, the initiator used for the IEP of DMA to UHMw PDMAis AIBN, ammonium persulfate, potassium persulfate, Michler's ketone,2-hydroxy-2-methyl-1-phenyl-propan-1-one,bis(2,6-dimethoxybenzoyl)-2,4,4-trimethylpentylphosphine oxide, or amixture thereof.

The inverse emulsion and/or its aqueous phase may also contain suchother additives if desired. These include chelating agents for removingpolymerization inhibitors, chain transfer agents, pH adjusters,co-initiators, sensitizers, charge-transfer complexes or donor-acceptorcomplexes when photoinitiation is used, and other conventional additivesused in their usual proportions. Polymerization in the inverse emulsionor microemulsion may be carried out by any manner known to those skilledin the art, e.g., as generally described in Griffin, pp. 919-923; U.S.Pat. No. 5,530,069 to Neff et al., at col. 3, lines 39-65 and col. 5,line 29 to col. 6, line 44; and in the references cited therein.Furthermore, the IEP of DMA to UHMw PDMA is described in Examples6.1.1-6.1.3 herein.

The Mw of the PDMA, when it is used as poly(M₁), can vary widely. In oneembodiment, the Mw of the PDMA used as poly(M₁) is from about 3 to about20 MDa when UHMw PDMA is prepared by IEP. In another embodiment, the Mwof an inverse emulsion polymerized UHMw PDMA poly(M₁) is from at leastabout 3 to about 10 MDa. In another embodiment, the present inventionrelates to the UHMw PDMA product of any of the methods herein for makingit. In another embodiment, the present invention relates to the UHMwPDMA product of any of the IEP methods herein for making it.

UHMw PDMA homopolymer (non-graft copolymer), i.e., not includingpoly(M₂), is also effective, e.g., in a CE separation medium forseparating biomolecules. Thus, a second embodiment of the inventionrelates to poly(N,N-dimethylacrylamide) where the weight-averagemolecular weight of the poly(N,N-dimethylacrylamide) is at least about 3MDa. In another embodiment, the sieve polymer ispoly(N,N-dimethyl-acrylamide) with a weight-average molecular weight ofat least about 3 MDa. In another embodiment, the UHMw PDMA has aweight-average molecular weight of from about 3 MDa to about 10 MDa.Additionally, a third embodiment of the invention relates to a methodfor making ultra-high molecular weight poly(N,N-dimethylacrylamide),comprising the step of polymerizing DMA in an inverse emulsioncomprising an oil phase, an aqueous phase, a surfactant and aninitiator, to provide poly(N,N-dimethylacrylamide) with a weight-averagemolecular weight of at least about 3 MDa. Another embodiment of theinvention relates to the poly(N,N-dimethylacrylamide) product of thismethod.

5.4 Method for Making Graft Copolymer (Continued)

Once a polymeric backbone has been obtained or prepared, apendant-forming polymerization reaction may be carried out, e.g., byfree-radical grafting with monomer or monomers M₂ as discussed above,thereby forming a graft copolymer of the invention. Without being boundby a particular theory, a proposed mechanism for free-radical graftingof an exemplary poly(M₁-g-M₂) of the invention, poly(DMA-g-AAm), isshown in the following scheme. Free-radicals 1, formed, for example, bythe thermal or photolytic decomposition of a free-radical initiator atthe start of polymerization, may initiate the polymerization of AAm 2 toform propagating macro-radical 3:

Free-radical 1 or macro-radical 3 can abstract a hydrogen atom from PDMA4 to form macro-radicals 5 and/or 6:

A termination reaction by coupling of 3 and 5 or 6 provides graftpoly(DMA-g-AAm) 7 having PAAm pendant(s) attached to a PDMA backbone:

Alternatively, macro-radicals 5 or 6 could initiate the polymerizationof AAm 2 to form a propagating PAAm pendant chain (not shown) grafted tothe PDMA backbone. However, without being bound by a particular theory,it is thought that macro-radicals 5 and 6 are each ineffective as aninitiator for the polymerization of 2.

Upon termination, e.g., by disproportionation, poly(DMA-g-AAm) 7 isobtained. If termination were to be by coupling between two suchpropagating PAAm pendant chains, crosslinking (not shown) could beexpected. However, based on the results reported in Example 6.2 belowfor example, any substantial amount of crosslinking does not occurbecause the poly(DMA-g-AAm) product prepared therein is water-soluble.

A fourth embodiment of the invention relates to a method for makingpoly(M₁-g-M₂), comprising the step of polymerizing M₂ in the presence ofpoly(M₁) to provide poly(M₁-g-M₂). It is to be understood that poly(M₁)can be a homopolymer or a copolymer. The polymerization may be initiatedby at least one free-radical, anionic and/or cationic initiator. Inanother embodiment, the polymerization is initiated by at least onefree-radical initiator. The free-radical initiator may be dissociatedthermally or photolytically in initiating the polymerization. In anotherembodiment, the poly(M₁-g-M₂) product has a weight-average molecularweight of from about 150,000 Da to about 20 MDa. In another embodiment,the present invention relates to the poly(M_(1-g)-M₂) product of any ofthe methods herein for making it.

In another embodiment of a method for making poly(M₁-g-M₂), the poly(M₁)is poly(N,N-dimethylacrylamide) and M₂ is acrylamide. In anotherembodiment, the free-radical initiator(s) are thermally orphotolytically dissociated initiators selected from azo compounds, diazocompounds, organic peroxides, organic hydroperoxides, organicpersulfates, organic hydropersulfates, inorganic peroxides, inorganicpersulfates, peroxide-redox systems, carbon-carbon initiators,photoinitiators, or a mixture thereof. Here, the PDMA backbone can havea Mw of from about 100,000 Da to about 10 MDa. Thus, the polymericbackbone can be solution-polymerized PDMA and have a Mw of from about500,000 Da to less than about 3 MDa, or it can be the inverseemulsion-polymerized UHMw PDMA of the invention and have a Mw of fromgreater than about 3 MDa to about 10 MDa.

Of course, other ways for initiating polymerization known in the art canalso be used to make the poly(M₁-g-M₂) of the invention. For example,exposing a combination of the poly(M₁) and monomer(s) to electron beams,ultraviolet radiation, usually in the presence of a photoinitiator, andhigh energy ionizing radiation sources, such as γ-radiation from a ⁶⁰Coor ¹³⁷Cs source, α-particles, β-particles, fast neutrons and x-rays, cancause the generation of free-radicals and/or ions that, in turn,initiate graft polymerization. Sanchez et al., “Initiators(Free-Radical),” at 454-457; Sheppard et al., “Initiators,” inKirk-Othmer Encyc. of Chem. Technol., 3rd Ed., John Wiley & Sons, NewYork, 1981, Vol. 13, pp. 367-370. At least the three following radiationgrafting methods are conventional: (1) the “pre-irradiation” method, inwhich the backbone polymer is irradiated before interacting with themonomer(s), (2) the “mutual radiation grafting” method, in which thebackbone polymer and the monomer(s) are in contact while irradiationoccurs, and (3) the “peroxide” method, in which the backbone polymer isirradiated in the presence of air or oxygen before interacting with themonomer(s). Stannett et al., “Polymerization by High-Energy Radiation”in Comprehensive Polymer Science, Pergamon Press, Oxford, 1989, Vol. 4,Eastmond et al., Eds., p. 327-334. Alternatively, it is possible tosynthesize graft copolymers using group-transfer polymerization.Costello, p. 359.

Another conventional method for preparing graft copolymers that can beused to prepare the poly(M₁-g-M₂) of the invention is the use oftelechelic polymers, also known as “macromonomers”. Costello, pp.360-361. As used herein, the term “telechelic polymer” refers to apolymer or oligomer having at least one functional end-group capable offorming bonds with another molecule. For example, a telechelic polymerconsisting essentially of vinyl-terminated polymer may be suitably used.The terminal vinyl group of such a telechelic polymer can becopolymerized with a monomer or monomers, e.g., M₁ of the invention, toform a graft copolymer bearing, as pendant chains, the polymer of thetelechelic polymer. Particularly, the telechelic polymer can bevinyl-terminated PAAm which, when copolymerized with M₁ of theinvention, yields poly(M₁-g-acrylamide), i.e., acrylamide as M₂.

Many ways of using a telechelic polymer in copolymerization are known.For example, U.S. Pat. No. 6,214,958 B1 to Le-Khac et al., at col. 3,line 55 to col. 6, line 8, discloses several different methods forpreparing copolymers comprising a polymeric backbone and pendantpolymeric side-chains from one or more telechelic polymers, includingusing a batch process, semi-batch or semi-continuous process (i.e., twoor more separate additions of monomer(s)), single stage continuousprocess, and multi-stage continuous process.

Alternatively, the poly(M₁-g-M₂) of the present invention can beprepared from polymeric starting materials, a method that is alsoconventional in making graft copolymers. For example, exposure of acombination of poly(M₂) and poly(M₂) to an ionizing radiation source canlead to the formation of macro-radical intermediates, e.g., by hydrogenabstraction or carbon-carbon bond cleavage. Then, the macro-radicalintermediates can couple to form a single, e.g., grafted, copolymermolecule having increased molecular weight relative to the startingpolymers. Adler, Science, 141:321-323 (1963); McGinniss, “RadiationCuring,” in Kirk-Othmer Encyc. of Chem. Technol., 3rd Ed., John Wiley &Sons, New York, 1982, Vol. 19, p. 612. Additionally, reactive processingmethods, such as reactive extrusion, can be used to make graftcopolymers in situ during polymer processing operations performed with acombination of poly(M₁) and poly(M₂). Costello, p. 377.

5.5 Compositions of the Invention

A fifth embodiment of the invention relates to a composition comprisingpoly(M₁-g-M₂) or a salt thereof and a buffer. The composition is usefulas an electrophoresis separation medium. In one embodiment, thecomposition further comprises a sieve polymer or a salt thereof(described in further detail below). In another embodiment, thecomposition further comprises a denaturant. In the compositions, thepoly(M₁-g-M₂) or a salt thereof is such that:

-   (a) each M₁ has the formula (I):

-   -   where each A₁ is independently O, S or NX₁;    -   each of R₁, R₂, R₃ and R₄ is independently H, C₁-C₂₀ alkyl,        C₄-C₁₂ cycloalkyl, C₅-C₁₂ aryl, C₄-C₁₂ heteroaryl, —(C₁-C₂₀        alkyl)(C₅-C₁₂ aryl) or —(C₅-C₁₂ aryl)(C₁-C₂₀ alkyl);    -   each R₅ is independently C₁-C₂₀ alkyl, C₁-C₂₀ heteroalkyl,        C₄-C₁₂ cycloalkyl, C₄-C₁₂ heterocycloalkyl, C₅-C₁₂ aryl, C₄-C₁₂        heteroaryl, —(C₁-C₂₀ alkyl)(C₄-C₁₂ cycloalkyl), —(C₄-C₁₂        cycloalkyl)(C₁-C₂₀ alkyl), —(C₁-C₂₀ heteroalkyl)(C₄-C₁₂        cycloalkyl), —(C₄-C₁₂ cycloalkyl)(C₁-C₂₀ heteroalkyl), —(C₁-C₂₀        alkyl)(C₄-C₁₂ heterocycloalkyl), —(C₄-C₁₂        heterocycloalkyl)(C₁-C₂₀ alkyl), —(C₁-C₂₀ heteroalkyl)(C₄-C₁₂        heterocycloalkyl), —(C₄-C₁₂ heterocycloalkyl)(C₁-C₂₀        heteroalkyl), —(C₁-C₂₀ alkyl)(C₅-C₁₂ aryl), —(C₅-C₁₂        aryl)(C₁-C₂₀ alkyl), —(C₁-C₂₀ heteroalkyl)(C₅-C₁₂ aryl),        —(C₅-C₁₂ aryl)(C₁-C₂₀ heteroalkyl), —(C₁-C₂₀ alkyl)(C₄-C₁₂        heteroaryl), —(C₄-C₁₂ heteroaryl)(C₁-C₂₀ alkyl), —(C₁-C₂₀        heteroalkyl)(C₄-C₁₂ heteroaryl), —(C₄-C₁₂ heteroaryl)(C₁-C₂₀        heteroalkyl), —(C₁-C₄ alkyl)_(q)NH₂, —(C₁-C₄ alkyl)_(q)CONH₂,        —(C₁-C₄ alkyl)NHCONH₂, —(C₁-C₄ alkyl)NHCOH or —(C₁-C₄        alkyl)_(q)NHCOCH₃, where each q is 0 or 1; and    -   each X₁ is independently H, C₁-C₂₀ alkyl, C₄-C₁₂ cycloalkyl,        C₅-C₁₂ aryl, C₄-C₁₂ heteroaryl, —(C₁-C₂₀ alkyl)(C₅-C₁₂ aryl),        —(C₅-C₁₂ aryl)(C₁-C₂₀ alkyl), —(C₁-C₄ alkyl)_(q)NH₂, (C₁-C₄        alkyl)_(q)CONH₂, —(C₁-C₄ alkyl)NHCONH₂, —(C₁-C₄ alkyl)_(q)NHCOH        or —(C₁-C₄ alkyl)_(q)NHCOCH₃, where each q is 0 or 1;

-   (b) each M₂ has the formula (II):

-   -   where each A₂ is independently O, S or NX₂;    -   each of R₆, R₇, R₈ and R₉ is independently H, C₁-C₂₀ alkyl,        C₄-C₁₂ cycloalkyl, C₅-C₁₂ aryl, C₄-C₁₂ heteroaryl, —(C₁-C₂₀        alkyl)(C₅-C₁₂ aryl) or —(C₅-C₁₂ aryl)(C₁-C₂₀ alkyl);    -   each R₁₀ is independently H, C₁-C₂₀ alkyl, C₁-C₂₀ heteroalkyl,        C₄-C₁₂ cycloalkyl, C₄-C₁₂ heterocycloalkyl, C₅-C₁₂ aryl, C₄-C₁₂        heteroaryl, —(C₁-C₂₀ alkyl)(C₄-C₁₂ cycloalkyl), —(C₄-C₁₂        cycloalkyl)(C₁-C₂₀ alkyl), —(C₁-C₂₀ heteroalkyl)(C₄-C₁₂        cycloalkyl), —(C₄-C₁₂ cycloalkyl)(C₁-C₂₀ heteroalkyl), —(C₁-C₂₀        alkyl)(C₄-C₁₂ heterocycloalkyl), —(C₄-C₁₂        heterocycloalkyl)(C₁-C₂₀—(C₁-C₂₀ heteroalkyl)(C₄-C₁₂        heterocycloalkyl), —(C₄-C₁₂ heterocycloalkyl)(C₁-C₂₀        heteroalkyl), —(C₁-C₂₀ alkyl)(C₅-C₁₂ aryl), —(C₅-C₁₂        aryl)(C₁-C₂₀ alkyl), —(C₁-C₂₀ heteroalkyl)(C₅-C₁₂ aryl),        —(C₅-C₁₂ aryl)(C₁-C₂₀ heteroalkyl), —(C₁-C₂₀ alkyl)(C₄-C₁₂        heteroaryl), —(C₄-C₁₂ heteroaryl)(C₁-C₂₀ alkyl), —(C₁-C₂₀        heteroalkyl)(C₄-C₁₂ heteroaryl), —(C₄-C₁₂ heteroaryl)(C₁-C₂₀        heteroalkyl), —(C₁-C₄ alkyl)_(q)NH₂, —(C₁-C₄ alkyl)_(q)CONH₂,        —(C₁-C₄ alkyl)NHCONH₂, —(C₁-C₄ alkyl)NHCOH or —(C₁-C₄        alkyl)_(q)NHCOCH₃, where each q is 0 or 1; and    -   each X₂ is independently H, C₁-C₂₀ alkyl, C₄-C₁₂ cycloalkyl,        C₅-C₁₂ aryl, C₄-C₁₂ heteroaryl, —(C₁-C₂₀ alkyl)(C₅-C₁₂ aryl),        —(C₅-C₁₂ aryl)(C₁-C₂₀ alkyl), —(C₁-C₄ alkyl)_(q)NH₂, —(C₁-C₄        alkyl)_(q)CONH₂, —(C₁-C₄ alkyl)NHCONH₂, —(C₁-C₄ alkyl)_(q)NHCOH        or —(C₁-C₄ alkyl)_(q)NHCOCH₃, where each q is 0 or 1;

-   (c) provided that at least one M₁ is different from at least one M₂.

In another embodiment, M₁ is N-adamantyl-acrylamide,N-butoxymethyl-acrylamide, N-butyl-acrylamide, N-cyclohexyl-acrylamide,N,N-dibutyl-acrylamide, N-3-di(butyl)aminopropyl-acrylamide,N,N-diethyl-acrylamide, N-4,4-dimethoxybutyl-acrylamide,N,N-dimethyl-acrylamide, N-3-(dimethylamino)-propyl-acrylamide,N,N-dipropyl-acrylamide, N-dodecyl-acrylamide,N-2-ethylhexyl-acrylamide, N-isobornyl-acrylamide, N-methyl-acrylamide,N-methyl-, N-2,2-dimethoxyethyl-acrylamide,N-morpholinoethyl-acrylamide, N-octadecyl-acrylamide,N-propyl-acrylamide, N-3-(trimethylammonium)-propyl-acrylamide chloride,N-1,1,3-trimethylbutyl-acrylamide, N-adamantyl-methacrylamide,N-butoxymethyl-methacrylamide, N-butyl-methacrylamide,N-cyclohexyl-methacrylamide, N,N-dibutyl-methacrylamide,N-3-di(butyl)aminopropyl-methacrylamide, N,N-diethyl-methacrylamide,N-4,4-dimethoxybutyl-methacrylamide, N,N-dimethyl-methacrylamide,N-3-(dimethylamino)-propyl-methacrylamide, N,N-dipropyl-methacrylamide,N-dodecyl-methacrylamide, N-2-ethylhexyl-methacrylamide,N-isobornyl-methacrylamide, N-methyl-methacrylamide, N-methyl-,N-2,2-dimethoxyethyl-methacrylamide, N-morpholinoethyl-methacrylamide,N-octadecyl-methacrylamide, N-propyl-methacrylamide,N-3-(trimethylammonium)-propyl-methacrylamide chloride,N-1,1,3-trimethylbutyl-methacrylamide, or a mixture thereof.

In another embodiment, M₂ is acrylamide, N-acetyl-acrylamide,N-butoxymethyl-acrylamide, N-4,4-dimethoxybutyl-acrylamide,N-2,2-dimethoxyethyl-acrylamide, N-2-glycolic acid methyl esteracrylamide, N-2-hydroxyethyl-acrylamide, N-hydroxymethyl-acrylamide,N-methoxymethyl-acrylamide, N-3-methoxypropyl-acrylamide,N-methyl-acrylamide, N-methyl-, N-2,2-dimethoxyethyl-acrylamide,N-morpholinoethyl-acrylamide,N-2,2,2-trichloro-1-hydroxyethyl-acrylamide,N-tri(hydroxymethyl)-methyl-acrylamide, methacrylamide,N-acetyl-methacrylamide, N-butoxymethyl-methacrylamide,N-4,4-dimethoxybutyl-methacrylamide,N-2,2-dimethoxyethyl-methacrylamide, N-2-glycolic acid methyl estermethacrylamide, N-2-hydroxyethyl-methacrylamide,N-hydroxymethyl-methacrylamide, N-methoxymethyl-methacrylamide,N-3-methoxypropyl-methacrylamide, N-methyl-methacrylamide, N-methyl-,N-2,2-dimethoxyethyl-methacrylamide, N-morpholinoethyl-methacrylamide,N-2,2,2-trichloro-1-hydroxyethyl-methacrylamide,N-tri(hydroxymethyl)-methyl-methacrylamide, or a mixture thereof.

Without being bound by a particular theory, the effectiveness of thecompositions of the invention is thought to arise from their ability tosuppress or eliminate EOF in CE.

The weight fraction of poly(M₁-g-M₂) present in a composition of theinvention, based on the total weight of composition, is from about0.0001 to about 0.02. In another embodiment, the weight fraction ofpoly(M₁-g-M₂) present in a composition of the invention is from about0.001 to about 0.015. In another embodiment, the weight fraction ofpoly(M₁-g-M₂) present in a composition of the invention is from about0.001 to about 0.005.

5.5.1 Buffer

The present compositions comprise an buffer system for controlling pH.In one embodiment, the buffer is an aqueous buffer. In anotherembodiment, the buffer is a substantially dry buffer. In anotherembodiment, the buffer is a dry buffer. In another embodiment, thebuffer provides a buffered composition with a pH of from about 5 toabout 11. In another embodiment, the buffer provides a bufferedcomposition with a pH of from about 7 to about 10. Exemplary aqueousbuffers include aqueous solutions of organic acids, such as citric,acetic or formic acid; zwitterionics, such asN-tris(hydroxymethyl)-2-aminoethane sulfonic acid (“TES”),N,N-bis(2-hydroxyethyl)glycine (“BICINE”),2-(2-amino-2-oxoethyl)-amino)ethane sulfonic acid (“ACES”) orglycylglycine; inorganic acids, such as phosphoric acid; and organicbases, such as 2-amino-2-(hydroxymethyl)-1,3-propanediol (“TRIS”).Exemplary substantially dry buffers can be prepared from each of theabove aqueous buffers by substantially evaporating the water. Exemplarydry buffers can be prepared from each of the above aqueous buffers bycompletely evaporating the water.

Buffer concentration can vary widely, for example from about 1 mmol toabout 1 mol, and often about 20 mmol/liter of water is suitable.Exemplary buffer solutions for conventional CE applications include thefollowing: 0.1 M TRIS, 0.25 M boric acid, 7 M urea with a pH of about7.6 for single stranded polynucleotide separations; or 0.089 M TRIS,0.089 M boric acid, 0.005 M ethylenediamine tetraacetic acid (“EDTA”)for double stranded polynucleotide separations.

In another embodiment, the buffers include “GA” buffer, “TTE” buffer, ora mixture thereof. GA buffer comprises3-((2-hydroxy-1,1-bis(hydroxymethyl)ethyl))-amino)-1-propanesulfonicacid sodium salt (“TAPS”) and EDTA with from about 1 to about 4 mM ofEDTA present per 100 mM of TAPS such that the pH of the buffer is about8.0. TTE buffer comprises TRIS, TAPS and EDTA with about 1 mM of EDTApresent per 50 mM of TRIS plus 50 mM of TAPS such that the pH of thebuffer is about 8.4.

An effective concentration of aqueous buffer present in a composition ofthe invention is from about 10 mM to about 300 mM. In one embodiment,the effective concentration of aqueous buffer is from about 25 mM toabout 200 mM. In another embodiment, the concentration of aqueous bufferpresent in a composition of the invention is from about 50 mM to about100 mM.

5.5.2 Sieve Polymer

Optionally, the present compositions comprise a sieve polymer. Inanother embodiment, the present compositions comprise a sieve polymer.In CE, it is postulated that a primary mechanism of separation fordifferent sized biomolecules, e.g., polynucleotides, is grounded ontheir charge-to-frictional drag ratio. Thus, it is desirable that asieve polymer is present if, in the absence of the same, two or morebiomolecules would co-migrate in CE, i.e., move with about the samemobility. For the purposes of this application, a “sieve polymer” meansa polymer present in an amount effective to cause at least twocomponents of a sample mixture to migrate with different mobilities inCE.

Gelled or crosslinked polymers can be useful sieve polymers. In anotherembodiment, non-covalently-crosslinked sieve polymers are used,comprising hydroxyalkylcellulose, agarose, cellulose acetate,essentially linear PAAm and the like, as disclosed by, e.g., Bode, Anal.Biochem., 83:204-210 (1977); Bode, Anal. Biochem., 83:364-371 (1977);Bode, Anal. Biochem., 92:99-110 (1979); Hjerten et al., J. LiquidChromatography, 12:2471-2477 (1989); U.S. Pat. No. 5,126,021 toGrossman; and Tietz et al., Electrophoresis, 13:614-616 (1992).

When present in the compositions of the invention in one embodiment, thesieve polymer is one or more substantially uncrosslinked polymers. Inanother embodiment, the sieve polymer is one or more substantiallylinear polymers.

In another embodiment, the sieve polymer is water soluble at atmosphericpressure, a concentration of from about 0.01 to about 1 wt. %, and fromabout 20° C. to about 70° C., e.g., at 25° C.

In one embodiment, when a sieve polymer is present in the compositionsof the invention, the sieve polymer has a weight-average molecularweight of from about 100,000 Da to about 5 MDa. In another embodiment,the sieve polymer has a weight-average molecular weight of from about500,000 Da to about 2 MDa. In another embodiment, the sieve polymer,when present, has a Mw of from about 800,000 Da to about 2 MDa.

In one embodiment, the sieve polymer comprises a monomer unit that isacrylamide, N-acetyl-acrylamide, N-2-cyanoethyl-acrylamide,N,N-1,2-dihydroxyethylene-bis-acrylamide,N-4,4-dimethoxybutyl-acrylamide, N-2,2-dimethoxyethyl-acrylamide,N,N-dimethyl-acrylamide, N-2-glycolic acid methyl ester acrylamide,N-2-hydroxyethyl-acrylamide, N-hydroxymethyl-acrylamide,N-methoxymethyl-acrylamide, N-3-methoxypropyl-acrylamide,N-methyl-acrylamide, N-methyl-, N-2,2-dimethoxyethyl-acrylamide,N-morpholinoethyl-acrylamide,N-2,2,2-trichloro-1-hydroxyethyl-acrylamide,N-tri(hydroxymethyl)-methyl-acrylamide, methacrylamide,N-acetyl-methacrylamide, N-2-cyanoethyl-methacrylamide,N,N-1,2-dihydroxyethylene-bis-methacrylamide,N-4,4-dimethoxybutyl-methacrylamide,N-2,2-dimethoxyethyl-methacrylamide, N,N-dimethyl-methacrylamide,N-2-glycolic acid methyl ester methacrylamide,N-2-hydroxyethyl-methacrylamide, N-hydroxymethyl-methacrylamide,N-methoxymethyl-methacrylamide, N-3-methoxypropyl-methacrylamide,N-methyl-methacrylamide, N-methyl-, N-2,2-dimethoxyethyl-methacrylamide,N-morpholinoethyl-methacrylamide,N-2,2,2-trichloro-1-hydroxyethyl-methacrylamide,N-tri(hydroxymethyl)-methyl-methacrylamide, or a mixture thereof.

In addition, the sieve polymer can comprise or ispoly(hydroxymethylene), poly(oxyethylene), poly(oxypropylene),poly(oxyethylene-co-oxypropylene), poly(vinyl alcohol),poly(vinylpyrrolidone), poly(2-ethyl-2-oxazoline),poly(2-methyl-2-oxazoline),poly((2-ethyl-2-oxazoline)-co-(2-methyl-2-oxazoline)),poly(N-acetamidoacrylamide), poly(acryloxylurea), a water-solublepolysaccharide such as hydroxyethyl cellulose or hydroxymethylcellulose, or a mixture thereof.

In one embodiment, the sieve polymer includes an acrylamide monomerunit. In another embodiment, at least about 80 mol % of the sievepolymer's monomer units are acrylamide. In another embodiment, at leastabout 90 mol % of the sieve polymer's monomer units are acrylamide. Inanother embodiment, at least about 95 mol % of the sieve polymer'smonomer units are acrylamide. In another embodiment, the sieve polymeris poly(acrylamide) that is substantially linear, i.e., in which thereis an insignificant amount of branching.

In one embodiment, the sieve polymer includes a N,N-dimethylacrylamidemonomer unit. In another embodiment, at least about 80 mol % of thesieve polymer's monomer units are N,N-dimethylacrylamide. In anotherembodiment, at least about 90 mol % of the sieve polymer's monomer unitsare N,N-dimethylacrylamide. In another embodiment, at least about 95 mol% of the sieve polymer's monomer units are N,N-dimethylacrylamide. Inanother embodiment, the sieve polymer is poly(N,N-dimethylacrylamide)that has a weight-average molecular weight of at least about 3 MDaltons.

When present, the weight fraction of sieve polymer in a composition ofthe invention, based on the total weight of all of the ingredientspresent in the composition, is from about 0.001 to about 0.1. In anotherembodiment, the weight fraction of sieve polymer in a composition of theinvention is from about 0.005 to about 0.05. In another embodiment, theweight fraction of sieve polymer present in a composition of theinvention is from about 0.01 to about 0.03 (0.01 wt. fraction=1 wt. %).

5.5.3 Denaturant

Additional optional components, such as denaturants, can be included inthe present compositions, e.g., when it is desirable to prevent theformation of duplexes or secondary structures in polynucleotides. In oneembodiment, denaturants include formamide, urea, detergents such assodium dodecyl sulfate, and commercially available lactams, such aspyrrolidone and N-methylpyrrolidone, as well as their mixtures. The useof denaturants in electrophoresis is conventional and is described in,e.g., recognized molecular biology references such as Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed. (Cold Spring HarborLaboratory, New York, 1989). In another embodiment, the denaturant, whenpresent, is formamide, urea, pyrrolidone, N-methylpyrrolidone or amixture thereof. In another embodiment, the denaturant, when present, isurea. In another embodiment, the denaturant, when present, is formamide.

When present, the concentration of denaturant in a composition of theinvention is from about 0.5 M to about 8 M. In another embodiment, theconcentration of denaturant is from about 2 M to about 8 M. In anotherembodiment, the concentration of denaturant present in a composition ofthe invention is from about 6 M to about 8 M.

A sixth embodiment of the invention relates to a composition comprisingUHMw PDMA and a buffer. The composition is useful as an electrophoresisseparation medium. In one embodiment, the composition further comprisesa sieve polymer or a salt thereof. In another embodiment, thecomposition further comprises a denaturant.

The weight fraction of UHMw PDMA present in a composition of theinvention, based on the total weight of all of the ingredients presentin the composition, is from about 0.0001 to about 0.02. In anotherembodiment, the weight fraction of UHMw PDMA present is from about 0.001to about 0.015. In another embodiment, the weight fraction of UHMw PDMApresent in a composition of the invention is from about 0.001 to about0.005. The amounts of buffer, sieve polymer when it is present, anddenaturant when it is present, are as given above.

5.6 Methods for Making Compositions

A seventh embodiment of the invention relates to a method for making acomposition from the poly(M₁-g-M₂), described in detail above, andcomprises contacting the poly(M₁-g-M₂) or a salt thereof with an aqueousbuffer, optionally also contacting with a sieve polymer or a saltthereof and/or a denaturant. The composition is useful as anelectrophoresis separation medium. For example, the composition may beprepared by dissolving, at 25° C., the polymeric components, includingthe poly(M₁-g-M₂) or salt thereof and, when it is present, the sievepolymer or salt thereof, in water followed by adding a concentrated formof the buffer. Alternatively, the poly(M₁-g-M₂) or salt thereof can bedissolved directly in the aqueous buffer and the optional sieve polymeradded to that solution. The denaturant can be present either before orafter the optional sieve polymer is added. Thus, the poly(M₁-g-M₂), andthe sieve polymer when it is present, can be added to water, aqueousbuffer, water plus denaturant, or aqueous buffer plus denaturant,depending on which combination is selected for use. Moreover, when thesieve polymer is present, it can be dissolved in, e.g., the buffer,before the poly(M₁-g-M₂) is introduced. Any suitable order of adding thecomponents for making such a composition comprising poly(M₁-g-M₂) iswithin the scope of this embodiment of the invention.

An eighth embodiment of the invention relates to a method for making acomposition from the UHMw PDMA, described in detail above, and comprisescontacting the UHMw PDMA with an aqueous buffer, optionally alsocontacting with a sieve polymer or a salt thereof and/or a denaturant.The composition is useful as an electrophoresis separation medium. Aspreviously discussed regarding the method for making a compositioncomprising poly(M₁-g-M₂), the order in which the contact occurs whenmaking a composition if the invention from UHMw PDMA is not critical.Thus, any order of adding the components for making such a compositionfrom UHMw PDMA is within the scope of this embodiment of the invention.

5.7 Methods for Separating

In a ninth embodiment of the invention, the compositions of theinvention are useful in a method for detecting or separating a sample oranalyte, e.g., a biomolecule or mixture of biomolecules. As used herein,“analyte” includes the substance for which a particular sample is beingtested, e.g., for the presence and/or amount contained in the sample.

For example, a suitable method for separating a mixture of biomoleculesusing a composition of the invention comprises:

-   (a) contacting a composition comprising poly(M₁-g-M₂) or a salt    thereof and a buffer with a mixture comprising a biomolecule; and-   (b) applying an electric field to the composition in an amount    sufficient to facilitate the separation of a biomolecule from the    mixture.

Another suitable method for separating a mixture of biomolecules using acomposition of the invention comprises:

-   (a) contacting a composition comprising UHMw PDMA and a buffer with    a mixture comprising a biomolecule; and-   (b) applying an electric field to the composition in an amount    sufficient to facilitate the separation of a biomolecule from the    mixture.

In another embodiment, the composition further comprises a sieve polymeror a salt thereof. In another embodiment, the composition furthercomprises a denaturant. In another embodiment, the composition is in asupport such as a capillary tube or column, prior to contacting with abiomolecule.

The biomolecule(s) can be a polynucleotide or polynucleotides. In oneembodiment, biomolecules include proteins, glycoproteins, natural andsynthetic peptides, alkaloids, polysaccharides, polynucleotides, and thelike. In another embodiment, biomolecule refers to polynucleotides.

The term “polynucleotide,” as used herein, refers to a linear polymer ofnatural or modified nucleoside monomers, including double and singlestranded deoxyribonucleosides, ribonucleosides, α-anomeric formsthereof, and the like. Usually the nucleoside monomers are linked byphosphodiester bonds or analogs thereof to form polynucleotides rangingin size from a few monomeric units, e.g., from about 8 to about 40, toseveral thousands of monomeric units. Whenever a polynucleotide isrepresented by a sequence of letters, such as “GTTACTG,” it will beunderstood that the nucleotides are in 5′→3′ order from left to rightand that “A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G”denotes deoxyguanosine, and “T” denotes thymidine, unless otherwisenoted. Analogs of phosphodiester linkages include phosphorothioate,phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like.

As used herein, “nucleoside” includes the natural nucleosides, including2′-deoxy and 2′-hydroxyl forms, e.g., as described in Komberg and Baker,DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992). “Analogs” inreference to nucleosides includes synthetic nucleosides having modifiedbase moieties and/or modified sugar moieties, e.g., described generallyby Scheit, Nucleotide Analogs (John Wiley, New York, 1980).

Regarding the degree of separation of a sample or analyte, it isconventional in CE that “resolution” or “Rs” is defined as:

Rs=0.59(X ₂ −X ₁)/FWHM  (1)

where X₁ and X₂ are the centers of two adjacent CE peaks and FWHM is thepeak width at half-height, assuming that both peaks have substantiallythe same width. (See, e.g., Albarghouthi, Electrophoresis, 21:4096-4111(2000)). As used herein, “crossover” is the base pair whose (X₂−X₁)value is equal to its FWHM value. In other words, a crossover of 650base pairs (“bp”) means that the resolution of the 650th base pair is0.59.

In one embodiment, a method for separating a mixture of biomoleculesusing a composition of the invention has a crossover of at least 400 bp.In another embodiment, a method for separating a mixture of biomoleculesusing a composition of the invention has a crossover of at least about600 bp. In another embodiment, a method for separating a mixture ofbiomolecules using a composition of the invention has a crossover of atleast about 700 bp. In another embodiment, a method for separating amixture of biomolecules using a composition of the invention has acrossover of at least about 800 bp. In another embodiment, a method forseparating a mixture of biomolecules using a composition of theinvention has a crossover of at least about 900 bp. In anotherembodiment, a method for separating a mixture of biomolecules using acomposition of the invention has a crossover of at least about 1000 bp.In another embodiment, a method for separating a mixture of biomoleculesusing a composition of the invention has a crossover of at least about1100 bp. In another embodiment, a method for separating a mixture ofbiomolecules using a composition of the invention has a crossover of atleast about 1200 bp.

Certain comparative separation media are known to undergo,disadvantageously, phase separation upon storage to yield two layers.Even remixing such phase-separated comparative separation media byeither shaking, tumbling or vigorous mechanical stirring can result in,e.g., significantly decreased crossover values when compared with theirpre-storage CE performance. However, it has been found that the storagestability of an electrophoresis separation medium can be improved bycontacting the separation medium with an effective amount of thepoly(M₁-g-M₂) of the invention. The weight fraction of poly(M₁-g-M₂)added to a phase-separated composition, based on the total weight of allof the ingredients present in the composition before the poly(M₁-g-M₂)is added, is from about 0.0001 to about 0.02, e.g., adding 0.18 wt % ofGC1 (as described in Example 6.2) restored the CE performance of aphase-separated comparative separation medium.

In contrast to the comparative separation media, which are known,disadvantageously, to undergo phase separation under certain storageconditions, the compositions of the invention are relatively stable,e.g., showing no significant change in DNA sequencing performance afterextended storage. For example, composition IC3 (as described in Example6.3) was a water-clear solution with no phase separation at the time ofits preparation and evaluation for DNA sequencing performance using CE.After aging for 3 months, the IC3 composition remained a water-clearsolution and no phase separation was observed. The after-aging CEperformance of IC3 was not significantly different from the samecomposition evaluated shortly after its preparation. Additionally, theafter-aging CE performance results of IC3 were highly reproducible overa number of runs. Furthermore, after aging for 6 months, composition IC2(as described in Example 6.3) remained a water-clear solution and nophase separation was observed. The after-aging CE performance of IC2 wasimproved, i.e., yielding a higher crossover at a lower run time, whencompared with the already good CE performance of the same compositionevaluated shortly after its preparation.

5.8 Electrophoresis Apparatus

A suitable electrophoresis apparatus comprises a support, e.g., acapillary, for a composition of the invention. In one embodiment, thesupport defines an elongate channel connectable at opposite ends toopposing polarity terminals of a voltage source and suitable forcontaining a composition of the invention, e.g., a separation medium.The term “capillary,” as used herein, refers to a tube or channel orother structure capable of supporting a volume of composition of theinvention for carrying out electrophoresis. The geometry of a support orcapillary may vary widely and includes tubes with circular, rectangularor square cross-sections, channels, groves, plates, and the like, eachof which may be fabricated by a wide range of technologies. For example,the support can comprise a CE array of bundled capillary channels.Alternately, the support can be of the microfabricated type, such as achannel chemically etched into a glass wafer as described by, e.g., Liuet al., Anal. Chem., 71:566-573 (1999). Exemplary references describingCE microchip apparatuses include the previous citation and Woolley etal., Anal. Chem., 67:3676-3680 (1995), which discloses a CE microchipwith a plurality of support channels, each measuring 50 μm wide and 8 μmdeep.

An important feature of a support suitable for use with the compositionof the invention is the surface area-to-volume ratio of the support'sinner surface in contact with the volume of the composition of theinvention. High values of this ratio permit better heat transfer fromthe composition during electrophoresis. In one embodiment, values in therange of from about 20,000 to about 200,000 are employed. Thesecorrespond to the surface area-to-volume ratios of tubular capillarieswith circular cross-sections having inside diameters of from about 200μm to about 10 μm.

In another embodiment, the support is a tubular capillary having alength of from about 10 to about 200 cm. In another embodiment, thesupport is a tubular capillary having a length of less than about 100cm. In another embodiment, the support is a tubular capillary having aninner diameter of from about 10 to about 200 μm. In another embodiment,the support is a tubular capillary having an inner diameter of fromabout 25 to about 75 μm.

Capillaries suitable for use with the invention may be made of silica,fused silica, silicate-based glass, such as borosilicate glass,alumina-containing glass, phosphate glass, quartz, and the like, orother silica-like materials. In one embodiment, the capillary comprisesfused silica. The capillary can be uncoated on its outside surface. Inanother embodiment, the capillary is coated on the outside surface witha polyimide layer, e.g., to provide suitable mechanical strengtheningand/or promote ease of handling. The capillary can be coated on itsinside surface, with one or a plurality of layers, typically with asilane-derived coating and/or PAAm as described in, e.g., U.S. Pat. No.4,997,537 to Karger et al., at col. 5, line 9 to col. 6, line 14. Inanother embodiment, the capillary is uncoated on the inside surface.

Apparatuses for performing capillary electrophoresis are well-known.Several CE instruments are commercially available, e.g., the AppliedBiosystems Inc. (ABI, Foster City, Calif.) model 310 CapillaryElectrophoresis Gene Analyzer. Exemplary references describing CEapparatus and their operation include Colburn et al., Applied BiosystemsResearch News, Issue 1 (Winter 1990); Grossman et al., Eds., CapillaryElectrophoresis (Academic Press, San Diego, 1992); Harrison et al.,Science, 261:895-897 (1993); U.S. Pat. No. 4,908,112 to Pace; U.S. Pat.No. 5,192,412 to Kambara et al.; and Seiler et al., Anal. Chem.,65:1481-1488 (1993).

Contacting a composition of the invention with the support such that thesupport contains the composition can be performed using conventionalmethods, e.g., by connecting one end to a syringe and injecting thecomposition into the support under a controlled pressure. When thesupport is a capillary, the injection pressure is suitably from about 50to about 800 psi. In another embodiment, the injection pressure for thecapillary support is from about 200 to about 400 psi. Alternately,contacting a composition of the invention with the support such that thesupport contains the composition can be performed by connecting thecapillary support to a filling tube and applying a nitrogen or heliumgas pressure of from about 100 to about 500 psi for from about 5 toabout 60 minutes, depending on the viscosity of the composition. U.S.Pat. No. 4,997,537 to Karger et al. discloses PAAm filling with a TEFLONtube and a syringe.

Another way for introducing a composition to the support so that thecomposition is contained therein is by immersing one of the two ends ofthe support into a reservoir containing a composition of the inventionand increasing the air pressure above that composition to greater thanatmospheric pressure, thereby forcing the composition into the supportvia positive pressure. Alternatively, the air pressure at the end of thesupport opposite to its immersed end may be reduced to belowatmospheric, thereby drawing the composition into the support bysuction.

Regardless of the method used, it is known in the art that the containedcomposition should fill the support substantially uniformly andhomogeneously, i.e., the composition should be substantially uniform indensity throughout the support and be substantially withoutdiscontinuities or voids. See, e.g., U.S. Pat. No. 5,468,365 to Menchenet al., col. 16, lines 33-45. The Brookfield viscosity of a compositionof the invention is suitably from about 100 to about 1000 cPs. Inanother embodiment, the Brookfield viscosity of a composition of theinvention is from about 200 to about 500 cPs. The compositions of theinvention are appropriately characterized by Method A of the ASTM D2196-99 test entitled “Standard Test Methods for Rheological Propertiesof Non-Newtonian Materials by Rotational (Brookfield type) Viscometer.”In this test and by “Method A” described therein, the apparent orBrookfield viscosity is determined by experimentally measuring thetorque on a spindle rotating at a constant speed within the liquidcomposition at a temperature of 25° C. Spindle No. 00 is used at arotational speed of 10 rpm in a Brookfield Model RV Viscometer or itsequivalent for all of these experiments.

A tenth embodiment of the invention relates to a support containing acomposition of the invention. In another embodiment, the support is acapillary. In another embodiment, the capillary is a tube.

In another embodiment, when multiple CE runs are conducted for a givencomposition/analyte combination, the composition is substantially, i.e.,99%, removed from the capillary at the completion of each CE run and afresh aliquot of the composition is introduced before the start of thenext CE run. In another embodiment, the entire removal and fillingoperation is conducted under automatic control, e.g., to promotereliable and reproducible CE results.

A cathodic reservoir can contain the composition into which a cathodeand the cathodic end of the capillary are immersed duringelectrophoresis, except for the brief period of time in which the sampleis introduced. The air pressure above the composition can be finelycontrolled, e.g., for loading the composition into the capillary bypositive pressure. An anodic reservoir can contain the composition intowhich an anode and the anodic end of the capillary is immersed duringelectrophoresis. The air pressure above that portion of the compositioncan also be finely controlled, if desired e.g., for drawing thecomposition into the capillary under reduced pressure. In anotherembodiment, the composition in the cathodic reservoir is substantiallyidentical to the composition in the anodic reservoir. The entire CEapparatus is maintained at a preselected constant temperature throughouta separation run.

A high voltage source is connected between the cathode and anode suchthat a constant run potential in the range of from about 2 to about 60kV is produced across the electrodes throughout CE. In anotherembodiment, the potential is in the range of from about 5 to about 20kV. Alternatively, or in addition, a selected-frequency pulsed voltagemay be applied between the electrodes, if desired. Currents through thecapillary during the CE run are suitably in the microamp range,typically from about 2 to about 100 μA. In another embodiment, currentsthrough the capillary during the CE run are from about 5 to about 30 μA.

A suitable sample or analyte to be analyzed using CE comprises abiomolecule or a mixture of such biomolecules. To begin a CE run, thesample may be introduced to the composition by any known means, e.g., bysyringe layering injection or differential pressure. In anotherembodiment, the sample is introduced by electrokinetic injection, e.g.,by placing the cathode and cathodic end of the capillary into a samplesolution then applying an injection potential and current across thecapillary for a short time. The sample is suitably electrokineticallyinjected for about 3 to about 150 seconds under a potential of fromabout 0.5 to about 18 kV. Separation commences after returning thecathode and cathodic end of the capillary into the cathode reservoir andapplication of the run potential and current.

An on-line detector positioned adjacent to capillary and nearer to itsanodic end monitors separated bands of sample migrating through adetection zone of the capillary. Typically, an optical detection zonecomprises a region of capillary in which any outer coating has beenremoved to permit UV and/or visible light, e.g. fluorescence, detectionof the separated analyte. However, a wide variety of detection schemesare amenable for use with the invention, including UV absorbance,fluorescence emission, laser-induced fluorescence, conductance,radioactive emission, and the like. For example, detection systems forfluorescent analytes are described in U.S. Pat. No. 4,675,300 to Zare etal. and U.S. Pat. No. 4,548,498 to Folestad et al. Alternately, a4-color detection system, such as is conventional in DNA analysis,utilizing an argon ion laser as a fluorescence-excitation light sourcethat emits light at wavelengths of 488 and 514 nm used in conjunctionwith a charged coupled device detector has be described in U.S. Pat. No.5,916,426 to Madabhushi et al.

Prior to its use with a different analyte and/or composition, thecapillary may be flushed, e.g., with 20 column volumes of water, 20column volumes tetrahydrofuran (THF), 20 column volumes 1 M NaOH and 20column volumes of water, before it is used to contain a fresh batch ofcomposition. In order to provide, e.g., reliable and reproducible CEresults, in one embodiment the used capillary is replaced with an unusedcapillary containing fresh composition and the sample is introduced byelectrokinetic injection, as described above.

6. EXAMPLES

As noted above, the graft copolymers and compositions and separationmedia containing the same; methods of making the graft copolymers andcompositions and separation media containing the same; and methods ofusing the graft copolymers and compositions and separation mediacontaining the same in CE yield superior CE performance in the analysisand separation of biomolecules. As also noted above, the UHMw PDMApolymers and compositions and separation media containing the same;methods of making the UHMw PDMA polymers and compositions and separationmedia containing the same; and methods of using the UHMw PDMA polymersand compositions and separation media containing the same in CE yieldsuperior CE performance in the analysis and separation of biomolecules.The following examples further illustrate certain embodiments of thepresent invention. These examples are provided solely for illustrativepurposes and in no way limit the scope of the present invention.

6.1.1 Preparation of UHMw Poly(N,N-dimethylacrylamide)

The following inverse emulsion polymerization method was used for thepreparation of ultra-high molecular weight poly(N,N-dimethylacrylamide)(“PDMA”).

A solution containing 12.022 g (121.28 mmol) of N,N-dimethylacrylamide(obtained from Monomer-Polymer & Dajac Laboratories, Inc., Feasterville,Pa., containing 75 ppm of 2,6-di-tert-butyl-4-methyl phenol), 4.9 mg(0.0132 mmol) of EDTA (99.999% obtained from Aldrich Chemical) and 6.1mg (0.021 mmol) of ammonium persulfate (99.99% obtained from AldrichChemical) in 18.0 g of distilled water was poured into a 500 mLpolypropylene beaker containing 80.0 g of mineral oil (certified forNujol mull and obtained from Aldrich Chemical) and 4.0 g of SPAN-80. Themixture was emulsified using a 2 inch rod-shape magnetic stir bar bystirring at 2,000 rpm for 5 minutes.

The resulting milky emulsion was transferred into a 500 mL three-neckedround bottom flask equipped with a mechanical stirrer having a 2 inchTEFLON stirring blade, a water-cooled condenser, a rubber septum, ableeding tube for bubbling and a 12 gauge syringe needle for venting.De-oxygenation of the emulsion was accomplished by bubbling in argon at130 mL/min for 1 hour with constant stirring at 200 rpm. Through therubber septum, 5 μL of N,N,N′,N′-tetramethylethylenediamine (“TEMED,”ultra-pure grade obtained from Armesco) was added with a syringe. Withconstant stirring at 200 rpm, the reaction flask was then immersed intoan oil bath maintained at 40±1° C. over a period of 22 hours. At the endof the reaction time, the oil bath was removed. With constant stirringat 200 rpm, air was bubbled into the reaction mixture for 5 minutes toquench the reaction.

The product mixture was divided into four 50 mL polyethylene centrifugetubes and each was centrifuged at 18,000 rpm and 15° C. for 30 minutes.The supernatant layer was decanted and the obtained pellets were rinsedwith n-hexane to remove the mineral oil. The resulting polymer wasvacuum dried at 35° C. overnight to produce 18.0 g of polymer.

To remove any residual mineral oil, a 5.6 g sample of the product wasadded to 150 mL of acetone and stirred to give a translucent solution.This acetone solution was poured in a fine stream into 1 L of n-hexanewith vigorous stirring. The precipitated polymer was rinsed withn-hexane and vacuum dried at 40° C. for 16 hours to provide 4.6 g ofPDMA, designated as UPDMA-1. The Mw of the UPDMA-1 was 8.0 MDa, asdetermined from batch mode MALLS testing.

6.1.2 Preparation of UHMw Poly(N,N-dimethylacrylamide)

The inverse emulsion polymerization method for the preparation of UHMwPDMA in Example 6.1.1 was carried out except for the followingmodifications. To 12.022 g of DMA was added a chilled solution of 4.2 mgof EDTA, 6.3 mg of ammonium persulfate and 18.0 g of distilled waterwith stirring. This solution was poured into a 250 mL beaker containing80.143 g of mineral oil and 1.05 g of TETRONIC 1301. The mixture wasemulsified using a 2 inch rod-shape magnetic stir bar for 10 minutes.

The resulting milky emulsion was transferred into a 250 mL three-neckedround bottom flask equipped as in Example 6.1.1. De-oxygenation of theemulsion was accomplished by bubbling in water-saturated argon for 75minutes. With constant stirring and purging with water-saturated argonat 130 mL/min, the reaction flask was then immersed into an oil bathmaintained at 50±1° C. over a period of 2 hours. At the end of thereaction time, the oil bath was removed and air was bubbled into thereaction mixture to quench the reaction.

To remove the residual monomer and mineral oil, the supernatant layerwas decanted, 200 mL of hexane was added, the obtained mixture wasstirred vigorously for 5 minutes and then centrifuged at 1,000 rpm for 2minutes. This procedure was repeated 3 times followed by a finaldecanting of the supernatant. The obtained polymer pellets werefreeze-dried to provide 11.50 g of polymer.

To further remove any residual mineral oil, a 1.007 g sample of theproduct was added to 20 mL of acetone in a 250 mL Erlenmeyer flask,stirred and dissolved to produce a water-clear solution. This acetonesolution was poured in a fine stream into 20 mL of vigorously stirredhexane to yield precipitated polymer, which was rinsed with excesshexane. The entire acetone-dissolution procedure above was thenrepeated. The resulting precipitated polymer was vacuum dried at 45° C.for 16 hours to produce 0.75 g of PDMA having a fibrous appearance.

For final purification, the above vacuum-dried polymer was placed in a125 mL polypropylene beaker containing 62 g of relatively pure waterobtained from a Dionics Corp. water purification system (conductivity ofabout 10 MΩ cm), stirred and dissolved to give a water-clear solution.This solution was filtered through a 5μ, ACRODISC syringe filter. Thefiltrate was freeze-dried as described above to produce a white polymerproduct having a fibrous appearance, designated as UPDMA-2. The Mw ofUPDMA-2 was determined as 6.1 MDa by GPC testing.

6.1.3 Preparation of UHMw Poly(N,N-dimethylacrylamide)

The inverse emulsion polymerization method for the preparation of UHMwPDMA in Example 6.1.2 was carried out except for the followingmodifications. To 12.02 g of DMA was added a chilled solution of 3.7 mgof EDTA and 18.0 g of distilled water with stirring. This solution waspoured into a 250 mL beaker containing 80.02 g of mineral oil and 4.01 gSPAN-80. The mixture was emulsified, transferred into a 250 mLthree-necked round bottom flask equipped as in Example 6.1.1, andde-oxygenated by bubbling in water-saturated argon for 60 minutes.

To the resulting de-oxygenated emulsion was added 6.2 mg of ammoniumpersulfate and 4 μL TEMED. With constant stirring and purging, thereaction flask was then immersed into an oil bath maintained at 42±1° C.over a period of 22 hours.

As described in Example 6.1.1, the resulting product mixture was dividedinto four centrifuge tubes and each was centrifuged at 18,000 rpm and10° C. for 30 minutes. The supernatant layer was decanted and theobtained pellets were briefly rinsed with hexane to remove the mineraloil. Then, the pellet in each centrifuge tube was redispersed with 40 mLof hexane and each dispersion was centrifuged again at 18,000 rpm and10° C. for 30 minutes. The supernatant was decanted and the resultingpolymer pellets were vacuum dried for 8 hours at 35° C.

To remove any residual mineral oil, a 3.5 g sample of the above polymerpellets was added to 75 mL of acetone and stirred to give a water-clearsolution. This acetone solution was poured into 500 mL ofvigorously-stirred hexane. The resulting precipitated polymer was rinsedwith hexane and vacuum dried to provide PDMA having a fibrousappearance, designated as UPDMA-3. The Mw of UPDMA-3 was determined as6.2 MDa by GPC testing (by American Polymer Standard Corporation,Mentor, Ohio).

6.2 Preparation of Poly(DMA-g-AAm) from Poly(N,N-dimethylacrylamide) andAcrylamide

Poly(dimethylacrylamide-g-acrylamide) (“poly(DMA-g-AAm)”) was preparedby the following representative procedure. A 500-mL three-necked roundbottom flask, equipped with a mechanical stirrer, a glass bleeding tubeand a syringe vent connected to an silicon oil bubbler, was charged with250.0 g of the relatively pure water obtained from the MILLI-Q WaterSystem (Millipore Corp., Bedford, Mass.), 7.0 g of a 28.57 wt % aqueoussolution of AAm monomer, and about 2 g of dialyzed, lypophilized PDMA.The PDMA was dialyzed with 50K MWCO Spectra/Por-7 regenerated cellulosemembranes for 4 days with two changes of water (5 gallons each) andlypophilized prior to its use. The PDMA weight and number-averagemolecular weight, as determined by GPC-MALLS was as follows: Mw=984,000Da and Mn=315,000 Da. The amount of AAm present in the above mixture wascalculated as about 2.0 g. To promote dissolution, the mixture wasstirred in open air.

To the water-clear solution, 0.43 mL of an aqueous solution of 1.98 wt %ammonium persulfate (8.5 mg) was added. The mixture was purged withultra-pure helium for 60 minutes at a flow rate of 150 mL/minute. At theend of the purging, 0.10 mL of 2-propanol was added. Table 1 summarizesthe materials used. The reaction mixture was then lowered into an oilbath maintained at 50±1° C. Polymerization was conducted at thattemperature over a period of 16 hours with constant stirring and underultra-pure helium bubbling in at a rate of 100 mL/minute.

TABLE 1 Formulations used for the preparation of poly(DMA-g-AAm) GraftAmmonium Copolymer AAm PDMA Approx. Water 2-Propanol persulfate Mw MnDesignation (g) (g) WFR* (g) (mL) (g) (MDa) (MDa) GC1 2.0011 2.0024 1250.0 0.10 0.0085 1.48 0.47 GC2 9.1654 0.9169 10 250.0 0.50 0.0396 2.151.02 * Weight Feed Ratio = (Weight monomer(s), g)/(Weight backbonepolymer, g)

After 16 hours, the reaction was stopped and the reaction mixture wasdialyzed as described above. After lyophilization, 3.7 g of the firstgraft copolymer (“GC1”) was obtained (92% yield) with the followingmolecular weight as determined by GPC-MALLS: Mw=1,477,000 Da, Mn=471,000Da.

A portion of this purified GC1 (1.93 g) was transferred into a Soxhletextractor and was extracted with boiling acetone continuously for 3 daysto remove any homopolymer of PDMA, which is known to be soluble inacetone. At the end of the extraction, the swollen polymer was vacuumdried for 5 hours at 40° C. to provide 1.42 g of extracted GC1 productthat was, because of these extreme extraction conditions, presumablyfree of PDMA homopolymer. GPC-MALLS determinations showed that theextracted GC1 graft polymer had the following properties: Mw=1,422,500Da and Mn=499,500 Da.

As illustrated in Table 1, a higher molecular weight graft copolymer 2(“GC2”) with a different AAm/PDMA ratio was prepared from the samestarting materials and using the above procedure except that the amountsof 2-propanol and ammonium persulfate and the feed ratio of AAm to PDMAwere varied.

While this example describes certain specific ingredients and methods,it should be recognized that satisfactory results are obtained byvarying the above-described procedure, for example, by using a chaintransfer agent other than 2-propanol. Additionally, combinations ofchain transfer agents can used. Similarly, an initiator other thanammonium persulfate can be used or combinations of initiators can beused.

6.3 Capillary Electrophoresis of DNA Using Poly(DMA-g-AAm)

Compositions comprising the graft copolymers of the present inventionwere evaluated for their suitability as capillary electrophoresisseparation media in DNA sequencing. In the following examples, eachcomposition and/or separation medium was evaluated in CE by using an ABI310 Capillary Electrophoresis Gene Analyzer equipped with a 47 cm longby 50 μm inner diameter uncoated fused silica capillary.

In each comparative separation medium, either GA buffer or TTE bufferwas used. Urea denaturant was also present. Each separation medium wasprepared by dissolving, at 25° C., the polymeric components in thebuffer plus denaturant.

CSM2 and 3 were each prepared with lyophilized, substantially linearPAAm of the following approximate molecular weight as determined byGPC-MALLS: Mw=1.5 MDa. CSM1 and 3 were each prepared with lyophilizedPDMA of the following approximate molecular weight as determined byGPC-MALLS: Mw=984,000 Da.

CE sequencing runs for these comparative separation media were conductedin the presence of a ladder of TET-dye labeled fragments, having lengthsof 35, 50, 75, 100, 139, 150, 160, 200, 250, 300, 340, 350, 400, 450,490, 500, 550, 600, 650 and 700 base pairs, at several temperatures,usually 50, 60 and 70° C., with 1.5 kV injection voltage and 10 secinjection time and with 9.5 kV run voltage. The crossover and run timewere determined at each temperature.

The composition and CE sequencing performance for each of thesecomparative separation media is summarized in Table 2.

TABLE 2 CE Crossover and Run Time of Comparative Separation Media,Average of 4 Runs Separation Run Time for 700 bp Medium Weight % UreaCrossover (bp) (min) Designation PAAm PDMA Buffer (wt %) 50° C. 60° C.70° C. 50° C. 60° C. 70° C. CSM1 — 4.9 GA 39.2 532 495 412 92.5 106.2137.3 CSM2 2.28 — GA 37.9 232 254 228 49.5 46.9 49.6

CSM1, lacking PAAm, had a low 50° C. crossover value of 532 bp and anexcessive run time (92.5 min). In CSM2, PDMA or any other dynamiccoating was absent. This formulation exhibited an extremely low 50° C.crossover value, 232 bp, the worst resolution of the three comparativeseparation media, but had a relatively short run time of 49.5 min.

Compositions of the invention were prepared from poly(DMA-g-AAm), e.g.,by the method described above but also using the dialyzed, lyophilized,extracted graft copolymer of this invention. The dialysis procedure wasas described above. IC1-3 were each prepared with the same PAAm used inCSM2. IC1 and 2 were each prepared with the graft copolymer GC2described in Example 6.2 while IC3 was prepared with theacetone-extracted graft copolymer GC1 described in that example. TET-dyelabeled fragment sequencing runs were conducted as described above forthe CSM separation media. The composition and CE sequencing performancefor each of the compositions of the invention is summarized in Table 3.In Tables 2 and 3, the weight percent values specified are based on thetotal weight of the composition, i.e., the separation medium.

TABLE 3 CE Crossover and Run Time of Compositions of the Invention,Average of 8 Runs Run Time for 700 bp Composition Weight % UreaCrossover (bp) (min) Designation PAAm GC2 GC1 Buffer (wt %) 50° C. 60°C. 70° C. 50° C. 60° C. 70° C. IC1 2.02 0.22 — GA 37.9 643 618 579 46.246.6 60.3 IC2 1.52 0.88 — GA 38.2 637 615 597 49.8 47.8 55.3 IC3 2.21 —0.20 GA 38.0 633 617 587 47.4 46.6 50.1

As illustrated in Table 3, IC1 was effective in CE. IC1 provided animproved crossover value at 50° C. of 643 bp, relative to the 532 bp ofCSM1 and the 232 bp of CSM2. At 50° C., IC1 had run times shorter thanCSM1 and CSM2.

As also illustrated in Table 3, decreasing the amount of PAAm (i.e., thesieve polymer) from 2.02 wt % in IC1 to 1.52 wt % in IC2, with acorresponding increase in the amount of graft copolymer from 0.22 to0.88 wt %, respectively, also resulted in a higher 50° C. crossovervalue relative to, e.g., CSM1, 637 bp to 532 bp, respectively. At 50°C., IC2 also had run times shorter than or comparable to CSM1 and CSM2.

A composition of the invention comprising the lower molecular weightgraft copolymer GC1, called IC3, was also effective in CE. IC3 providedan improved crossover value at 50° C. of 633 bp, relative to the 532 bpof CSM1 and the 232 bp of CSM2, also with shorter run times.

6.4 CE as a Confirmation of the Structure of the Poly(DMA-g-AAm)

Poly(DMA-g-AAm) graft copolymers of the invention were prepared usingfree-radical polymerization of acrylamide in an aqueous solution ofPDMA, as described in Example 6.2. After dialysis and lyophilization, asalso described therein, which removed unreacted acrylamide, initiatorand other low molecular weight impurities, only the following polymericproducts could have been present in the isolate: (1) poly(DMA-g-AAm);(2) PAAm, which is relatively acetone insoluble; and (3) unreacted PDMA,which is very acetone soluble.

As described in Example 6.2, the “purified GC1” was subjected to boilingacetone extraction to produce “extracted GC1”. Such extraction was usedbecause neither GC1 nor GC2 was soluble in acetone. Since PDMA is verysoluble in acetone and the purified GC1 of Example 6.2 was swollen inboiling acetone for 3 days of acetone extraction in the Soxhletextractor, an undetectable amount of PDMA remained in the extracted GC1.

The results from DNA sequencing using CE clearly distinguish amongseparation media containing only PAAm and those containing PDMA andPAAm. A separation medium containing PAAm as the sole polymericcomponent, i.e., CSM2 in Table 2 above, does not suppress EOF wellenough to give a high crossover value. In contrast, the extractedcopolymer products IC1-3 of Table 3, when present with PAAm incompositions of the invention used as a separation media, performedmarkedly better than CSM2, e.g., by effectively suppressing EOF asindicated by their high crossover values relative to CSM2. This greatlyimproved CE performance confirms that the PDMA reactant was chemicallyincorporated into the graft copolymers GC1 and GC2 present in IC1-3.

6.5 Capillary Electrophoresis of DNA Using UHMw PDMA

Capillary electrophoresis separation media IC4 and IC5, each comprisingan UHMw PDMA polymer of the present invention, were evaluated for theirsuitability for DNA sequencing.

IC4 was made from 3.6 wt % of the UPDMA-2 UHMw PDMA prepared by theprocedure of Example 6.1.2. IC5 was made from 2.6 wt % of the UPDMA-3UHMw PDMA prepared by the procedure of Example 6.1.3. GA buffer was usedand 39.2 wt % of urea denaturant was also present in each of IC4 andIC5. Each of these separation media was prepared by dissolving, at 25°C., the UHMw PDMA in the buffer plus denaturant.

IC4 was evaluated for DNA sequencing performance using CE as describedin Example 6.3. The results were as follows: a 50° C. crossover of 625bp with a 76 minute run time, each from an average of 4 runs. Thus, IC4,containing UPDMA-2 and lacking PAAm, had a much higher 50° C. crossovervalue of 625 bp when compared with the 532 bp obtained with CSM1,containing lower molecular weight PDMA and also lacking PAAm.Additionally, the 76 minute run time for IC4 at 50° C. was,advantageously, far shorter when compared to the 92.5 minute run time ofCSM1.

IC5 was also evaluated for DNA sequencing performance using CE asdescribed in Example 6.3. The results were as follows: a 50° C.crossover of 610 bp with a 52 minute run time, each from an average of 4runs. 105, containing UPDMA-3, also had a much higher 50° C. crossovervalue of 610 bp when compared with the 532 bp obtained with CSM1,containing lower molecular weight PDMA and also lacking PAAm.Additionally, the run time for 105, at 50° C., at 52 minutes, was alsodramatically and advantageously lower when compared to the 92.5 minuterun time of CSM1.

All publications and patent applications are herein incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference.

All concentrations herein are by weight unless otherwise noted.

Although the invention has been described with reference to particularembodiments, it will be appreciated that various changes andmodifications may be made without departing from the spirit or scope ofthe invention.

What is claimed is:
 1. A composition comprising a buffer and aneffective amount of a poly(M₁-g-M₂) or a salt thereof, wherein: (a) eachM₁ has the formula (I):

wherein each A₁ is independently O, S or NX₁; each of R₁, R₂, R₃ and R₄is independently H, C₁-C₂₀ alkyl, C₄-C₁₂ cycloalkyl, C₅-C₁₂ aryl, C₄-C₁₂heteroaryl, —(C₁-C₂₀ alkyl)(C₅-C₁₂ aryl) or —(C₅-C₁₂ aryl)(C₁-C₂₀alkyl); each R₅ is independently C₁-C₂₀ alkyl, C₁-C₂₀ heteroalkyl,C₄-C₁₂ cycloalkyl, C₄-C₁₂ heterocycloalkyl, C₅-C₁₂ aryl, C₄-C₁₂heteroaryl, —(C₁-C₂₀ alkyl)(C₄-C₁₂ cycloalkyl), —(C₄-C₁₂cycloalkyl)(C₁-C₂₀ alkyl), —(C₁-C₂₀ heteroalkyl)(C₄-C₁₂ cycloalkyl),—(C₄-C₁₂ cycloalkyl)(C₁-C₂₀ heteroalkyl), —(C₁-C₂₀ alkyl)(C₄-C₁₂heterocycloalkyl), —(C₄-C₁₂ heterocycloalkyl)(C₁-C₂₀ alkyl), —(C₁-C₂₀heteroalkyl)(C₄-C₁₂ heterocycloalkyl), —(C₄-C₁₂ heterocycloalkyl)(C₁-C₂₀heteroalkyl), —(C₁-C₂₀ alkyl)(C₅-C₁₂ aryl), —(C₅-C₁₂ aryl)(C₁-C₂₀alkyl), —(C₁-C₂₀ heteroalkyl)(C₅-C₁₂ aryl), —(C₅-C₁₂ aryl)(C₁-C₂₀heteroalkyl), —(C₁-C₂₀ alkyl)(C₄-C₁₂ heteroaryl), —(C₄-C₁₂heteroaryl)(C₁-C₂₀ alkyl), —(C₁-C₂₀ heteroalkyl)(C₄-C₁₂ heteroaryl),—(C₄-C₁₂ heteroaryl)(C₁-C₂₀ heteroalkyl), —(C₁-C₄ alkyl)_(q)NH₂, —(C₁-C₄alkyl)_(q)CONH₂, —(C₁-C₄ alkyl)NHCONH₂, —(C₁-C₄ alkyl)NHCOH or —(C₁-C₄alkyl)_(q)NHCOCH₃, where each q is 0 or 1; and each X₁ is independentlyH, C₁-C₂₀ alkyl, C₄-C₁₂ cycloalkyl, C₅-C₁₂ aryl, C₄-C₁₂ heteroaryl,—(C₁-C₂₀ alkyl)(C₅-C₁₂ aryl), —(C₅-C₁₂ aryl)(C₁-C₂₀ alkyl), —(C₁-C₄alkyl)_(q)NH₂, —(C₁-C₄ alkyl)_(q)CONH₂, —(C₁-C₄ alkyl)NHCONH₂, —(C₁-C₄alkyl)_(q)NHCOH or —(C₁-C₄ alkyl)_(q)NHCOCH₃, where each q is 0 or 1;(b) each M₂ has the formula (II):

wherein each A₂ is independently O, S or NX₂; each of R₆, R₇, R₈ and R₉is independently H, C₁-C₂₀ alkyl, C₄-C₁₂ cycloalkyl, C₅-C₁₂ aryl, C₄-C₁₂heteroaryl, —(C₁-C₂₀ alkyl)(C₅-C₁₂ aryl) or —(C₅-C₁₂ aryl)(C₁-C₂₀alkyl); each R₁₀ is independently H, C₁-C₂₀ alkyl, C₁-C₂₀ heteroalkyl,C₄-C₁₂ cycloalkyl, C₄-C₁₂ heterocycloalkyl, C₅-C₁₂ aryl, C₄-C₁₂heteroaryl, —(C₁-C₂₀ alkyl)(C₄-C₁₂ cycloalkyl), —(C₄-C₁₂cycloalkyl)(C₁-C₂₀ alkyl), —(C₁-C₂₀ heteroalkyl)(C₄-C₁₂ cycloalkyl),—(C₄-C₁₂ cycloalkyl)(C₁-C₂₀ heteroalkyl), —(C₁-C₂₀ alkyl)(C₄-C₁₂heterocycloalkyl), —(C₄-C₁₂ heterocycloalkyl)(C₁-C₂₀ alkyl), —(C₁-C₂₀heteroalkyl)(C₄-C₁₂ heterocycloalkyl), —(C₄-C₁₂ heterocycloalkyl)(C₁-C₂₀heteroalkyl), —(C₁-C₂₀ alkyl)(C₅-C₁₂ aryl), —(C₅-C₁₂ aryl)(C₁-C₂₀alkyl), —(C₁-C₂₀ heteroalkyl)(C₅-C₁₂ aryl), —(C₅-C₁₂ aryl)(C₁-C₂₀heteroalkyl), —(C₁-C₂₀ alkyl)(C₄-C₁₂ heteroaryl), —(C₄-C₁₂heteroaryl)(C₁-C₂₀ alkyl), —(C₁-C₂₀ heteroalkyl)(C₄-C₁₂ heteroaryl),—(C₄-C₁₂ heteroaryl)(C₁-C₂₀ heteroalkyl), —(C₁-C₄—(C₁-C₄alkyl)_(q)CONH₂, —(C₁-C₄ alkyl)NHCONH₂, —(C₁-C₄ alkyl)NHCOH or —(C₁-C₄alkyl)_(q)NHCOCH₃, where each q is 0 or 1; and each X₂ is independentlyH, C₁-C₂₀ alkyl, C₄-C₁₂ cycloalkyl, C₅-C₁₂ aryl, C₄-C₁₂ heteroaryl,—(C₁-C₂₀ alkyl)(C₅-C₁₂ aryl), —(C₅-C₁₂ aryl)(C₁-C₂₀ alkyl), —(C₁-C₄alkyl)_(q)NH₂, —(C₁-C₄ alkyl)_(q)CONH₂, —(C₁-C₄ alkyl)NHCONH₂, —(C₁-C₄alkyl)_(q)NHCOH or —(C₁-C₄ alkyl)_(q)NHCOCH₃, where each q is 0 or 1;(c) provided that at least one M₁ is different from at least one M₂. 2.The composition of claim 1, which further comprises a sieve polymer, ora salt thereof, having a monomer unit that is acrylamide,N-acetyl-acrylamide, N-2-cyanoethyl-acrylamide,N,N-1,2-dihydroxyethylene-bis-acrylamide,N-4,4-dimethoxybutyl-acrylamide, N-2,2-dimethoxyethyl-acrylamide,N,N-dimethyl-acrylamide, N-2-glycolic acid methyl ester acrylamide,N-2-hydroxyethyl-acrylamide, N-hydroxymethyl-acrylamide,N-methoxymethyl-acrylamide, N-3-methoxypropyl-acrylamide,N-methyl-acrylamide, N-methyl-, N-2,2-dimethoxyethyl-acrylamide,N-morpholinoethyl-acrylamide,N-2,2,2-trichloro-1-hydroxyethyl-acrylamide,N-tri(hydroxymethyl)-methyl-acrylamide, methacrylamide,N-acetyl-methacrylamide, N-2-cyanoethyl-methacrylamide,N,N-1,2-dihydroxyethylene-bis-methacrylamide,N-4,4-dimethoxybutyl-methacrylamide,N-2,2-dimethoxyethyl-methacrylamide, N,N-dimethyl-methacrylamide,N-2-glycolic acid methyl ester methacrylamide,N-2-hydroxyethyl-methacrylamide, N-hydroxymethyl-methacrylamide,N-methoxymethyl-methacrylamide, N-3-methoxypropyl-methacrylamide,N-methyl-methacrylamide, N-methyl-, N-2,2-dimethoxyethyl-methacrylamide,N-morpholinoethyl-methacrylamide,N-2,2,2-trichloro-1-hydroxyethyl-methacrylamide,N-tri(hydroxymethyl)-methyl-methacrylamide, or a mixture thereof.
 3. Thecomposition of claim 2, wherein the sieve polymer is poly(acrylamide).4. The composition of claim 2, wherein the sieve polymer ispoly(N,N-dimethyl-acrylamide) and the sieve polymer has a weight-averagemolecular weight of at least about 3 MDaltons.
 5. A method for makingpoly(N,N-dimethylacrylamide), the method comprising polymerizingN,N-dimethylacrylamide in an inverse emulsion comprising an oil phase,an aqueous phase, a surfactant and an initiator to provide thepoly(N,N-dimethylacrylamide), wherein the poly(N,N-dimethylacrylamide)has a weight-average molecular weight of at least about 3 MDaltons. 6.The method of claim 5, wherein the oil phase comprises an aliphatichydrocarbon having at least about 15 carbon atoms, an aliphatichydrocarbon having a normal boiling point at or above about 270° C., asilicone oil, a fluorinated hydrocarbon, a liquid perfluoropolyether, ora mixture thereof.
 7. The poly(N,N-dimethylacrylamide) product of themethod of claim
 5. 8. The composition of claim 1, which furthercomprises poly(hydroxymethylene), poly(oxyethylene), poly(oxypropylene),poly(oxyethylene-co-oxypropylene), polyvinyl alcohol),poly(vinylpyrrolidone), poly(2-ethyl-2-oxazoline),poly(2-methyl-2-oxazoline),poly((2-ethyl-2-oxazoline)-co-(2-methyl-2-oxazoline)),poly(N-acetamidoacrylamide), poly(acryloxylurea), hydroxyethylcellulose, hydroxymethyl cellulose, or a mixture thereof.
 9. Thecomposition of claim 1, wherein the poly(M₁-g-M₂) or a salt thereof hasa weight-average molecular weight of from about 150,000 Daltons to about20 MDaltons.
 10. The composition of claim 9, which further comprises asieve polymer or a salt thereof having a weight-average molecular weightof from about 100,000 Daltons to about 5 MDaltons.
 11. The compositionof claim 10, wherein the sieve polymer is substantially linearpoly(acrylamide).
 12. The composition of claim 1, wherein M₁ is:N-adamantyl-acrylamide, N-butoxymethyl-acrylamide, N-butyl-acrylamide,N-cyclohexyl-acrylamide, N,N-dibutyl-acrylamide,N-3-di(butyl)aminopropyl-acrylamide, N,N-diethyl-acrylamide,N-4,4-dimethoxybutyl-acrylamide, N,N-dimethyl-acrylamide,N-3-(dimethylamino)-propyl-acrylamide, N,N-dipropyl-acrylamide,N-dodecyl-acrylamide, N-2-ethylhexyl-acrylamide, N-isobornyl-acrylamide,N-methyl-acrylamide, N-methyl-, N-2,2-dimethoxyethyl-acrylamide,N-morpholinoethyl-acrylamide, N-octadecyl-acrylamide,N-propyl-acrylamide, N-3-(trimethylammonium)-propyl-acrylamide chloride,N-1,1,3-trimethylbutyl-acrylamide, N-adamantyl-methacrylamide,N-butoxymethyl-methacrylamide, N-butyl-methacrylamide,N-cyclohexyl-methacrylamide, N,N-dibutyl-methacrylamide,N-3-di(butyl)aminopropyl-methacrylamide, N,N-diethyl-methacrylamide,dimethoxybutyl-methacrylamide, N,N-dimethyl-methacrylamide,N-3-(dimethylamino)-propyl-methacrylamide, N,N-dipropyl-methacrylamide,N-dodecyl-methacrylamide, N-2-ethylhexyl-methacrylamide,N-isobornyl-methacrylamide, N-methyl-methacrylamide, N-methyl-,N-2,2-dimethoxyethyl-methacrylamide, N-morpholinoethyl-methacrylamide,N-octadecyl-methacrylamide, N-propyl-methacrylamide,N-3-(trimethylammonium)-propyl-methacrylamide chloride,N-1,1,3-trimethylbutyl-methacrylamide, or a mixture thereof.
 13. Thecomposition of claim 12, wherein M₂ is: acrylamide, N-acetyl-acrylamide,N-butoxymethyl-acrylamide, N-4,4-dimethoxybutyl-acrylamide,N-2,2-dimethoxyethyl-acrylamide, N-2-glycolic acid methyl esteracrylamide, N-2-hydroxyethyl-acrylamide, N-hydroxymethyl-acrylamide,N-methoxymethyl-acrylamide, N-3-methoxypropyl-acrylamide,N-methyl-acrylamide, N-methyl-, N-2,2-dimethoxyethyl-acrylamide,N-morpholinoethyl-acrylamide,N-2,2,2-trichloro-1-hydroxyethyl-acrylamide,N-tri(hydroxymethyl)-methyl-acrylamide, methacrylamide,N-acetyl-methacrylamide, N-butoxymethyl-methacrylamide,N-4,4-dimethoxybutyl-methacrylamide,N-2,2-dimethoxyethyl-methacrylamide, N-2-glycolic acid methyl estermethacrylamide, N-2-hydroxyethyl-methacrylamide,N-hydroxymethyl-methacrylamide, N-methoxymethyl-methacrylamide,N-3-methoxypropyl-methacrylamide, N-methyl-methacrylamide, N-methyl-,N-2,2-dimethoxyethyl-methacrylamide, N-morpholinoethyl-methacrylamide,N-2,2,2-trichloro-1-hydroxyethyl-methacrylamide,N-tri(hydroxymethyl)-methyl-methacrylamide, or a mixture thereof. 14.The composition of claim 12, wherein M₂ is: N-acetyl-acrylamide,N-butoxymethyl-acrylamide, N-4,4-dimethoxybutyl-acrylamide,N-2,2-dimethoxyethyl-acrylamide, N-2-glycolic acid methyl esteracrylamide, N-2-hydroxyethyl-acrylamide, N-hydroxymethyl-acrylamide,N-methoxymethyl-acrylamide, N-3-methoxypropyl-acrylamide,N-methyl-acrylamide, N-methyl-, N-2,2-dimethoxyethyl-acrylamide,N-morpholinoethyl-acrylamide,N-2,2,2-trichloro-1-hydroxyethyl-acrylamide,N-tri(hydroxymethyl)-methyl-acrylamide, N-acetyl-methacrylamide,N-butoxymethyl-methacrylamide, N-4,4-dimethoxybutyl-methacrylamide,N-2,2-dimethoxyethyl-methacrylamide, N-2-glycolic acid methyl estermethacrylamide, N-2-hydroxyethyl-methacrylamide,N-hydroxymethyl-methacrylamide, N-methoxymethyl-methacrylamide,N-3-methoxypropyl-methacrylamide, N-methyl-methacrylamide, N-methyl-,N-2,2-dimethoxyethyl-methacrylamide, N-morpholinoethyl-methacrylamide,N-2,2,2-trichloro-1-hydroxyethyl-methacrylamide,N-tri(hydroxymethyl)-methyl-methacrylamide, or a mixture thereof. 15.The composition of claim 1, wherein the buffer is an aqueous buffer. 16.The composition of claim 15, wherein the composition has a pH of fromabout 5 to about
 11. 17. The composition of claim 15, wherein thecomposition has a pH of from about 7 to about
 10. 18. The composition ofclaim 15, wherein M₁ is N,N-dimethylacrylamide and M₂ is acrylamide. 19.The composition of claim 16, further comprising formamide, urea,pyrrolidone, N-methylpyrrolidone or a mixture thereof.
 20. Thecomposition of claim 16, further comprising urea.
 21. The composition ofclaim 16, further comprising formamide.
 22. A capillary containing thecomposition of claim
 1. 23. The capillary of claim 22, wherein thecapillary is a capillary tube.
 24. A method for separating a mixture ofbiomolecules, comprising: (a) contacting the composition of claim 1 witha mixture comprising a biomolecule; and (b) applying an electric fieldto the composition in an amount sufficient to facilitate the separationof a biomolecule from the mixture.
 25. The method of claim 24, whereinthe separation is performed within a capillary tube and two or morebiomolecules are polynucleotides.
 26. The method of claim 25, whereinthe separation has a crossover of at least 400 base pairs. 27.Poly(N,N-dimethylacrylamide) having a weight-average molecular weight ofat least about 3 MDaltons.